Mechano-activated control of gene expression

ABSTRACT

Described herein are compositions, methods, and systems for modulating Notch receptor activation. Aspects of the invention relate to synthetic proteins comprising at least a Notch NRR (Negative Regulatory Region)-binding scFV fused to a transmembrane domain. Another aspect of the invention relates to drug-dependent synthetic proteins. Constructs and engineered cells comprising the synthetic proteins are additionally described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation under 35 U.S.C. § 120 of co-pending U.S. application Ser. No. 15/994,330 filed May 31, 2018, which claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 62/513,031 filed May 31, 2017 and U.S. Provisional Application No. 62/586,451 filed Nov. 15, 2017, the contents of each of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 29, 2018, is named 701586-089500USPT_SL.txt and is 208,433 bytes in size.

TECHNICAL FIELD

The present invention relates generally to compositions and methods for engineering natural and synthetic Notch signaling.

BACKGROUND OF THE INVENTION

Drug-inducible strategies for regulating protein function and gene activity have been indispensable tools in biological research, yet methods for controlling diverse systems remain lacking. The Notch protein is a transmembrane receptor that acts a mechanical “switch,” translating mechanical cues into gene expression. This mechanosensing activity is achieved via Notch's force-sensitive Negative Regulatory Region (NRR), which contains three LNR domains. In the resting state, the LNR domains adopt an autoinhibitory conformation that sterically hinders proteolytic cleavage necessary for receptor activation. Upon the application of a pulling force, however, these LNR domains are displaced, and two concomitant proteolytic cleavages occur that release the Notch intracellular domain to transport to the nucleus and regulate gene expression.

SUMMARY OF THE INVENTION

As described herein, compositions, methods, and systems using antibody domains have been developed through which signaling from natural and synthetic Notch receptors can be regulated or modulated. These compositions, methods, and systems can be used to increase the amount of force required to activate Notch receptors, or to regulate their activity on therapeutic cells. The compositions, methods, and systems described herein are useful for a variety of cell engineering applications, including the creation of engineered cells capable of sensing certain mechanical features of solid tumors (or biomaterials), as well as for precisely controlling therapeutic and/or engineered cells expressing synthetic receptor proteins, such as synthetic Notch receptor proteins.

The compositions, methods, and systems described herein involve, in part, the use of antibody fragments directed against the NRR region of the Notch receptor, a force-sensitive mechanical switch that is ruptured during receptor activation. Binding of these antibodies stabilizes the NRR and prevents Notch activation. Use of scFvs from these antibodies permits the generation of inhibitory “modules” which are used to generate synthetic proteins and receptors for precisely controlling signaling from Notch and synthetic Notch systems. For example, synthetic notch receptors have been created that allow for gene expression to be controlled upon binding of the receptor to various ligands of interest (e.g., surface proteins on cancer cells), such as those described in US20160264665, the contents of which are herein incorporated by reference in their entireties.

As described herein, antibody fragments from anti-NRR antibodies are used as modules for engineering synthetic proteins and receptors for cell-engineering applications. These antibody-derived fragments are used as new genetic tools to reprogram natural and synthetic Notch signaling for synthetic biology applications. In some embodiments, these can be used to allow cells expressing these systems to function as genetically encoded “tensometers,” permitting, for example, engineered T cells to activate their cell killing activity in response to the mechanical properties of fibrotic tissues or physical features of solid tumors. In contrast, previous work involving anti-NRR antibodies relied on the use of purified immunoglobulin as an exogenously applied drug or agent.

The compositions, methods, and systems described herein are also directed towards compositions, constructs, and methods for controlling the binding/activation of Notch receptors through the use of synthetic protein ligands that incorporate NS3 protease domains as a Ligand-Inducible Connection (LInC) from the hepatitis C virus. In the absence of an NS-inhibitor drug, the ligand domain is cleaved and becomes incapable of activating the notch receptor. When an NS-inhibitor drug is applied, the NS3 domain remains intact and the ligand is capable of activating the notch receptor. As demonstrated herein, using the NS3 cis-protease from hepatitis C virus (HCV) as a LInC or drug-inducible linker allows precise control of protein function and localization using clinically tested antiviral protease inhibitors. The versatility of the approach is demonstrated in the design of drug-sensitive transcription factors (TFs) and transmembrane signaling proteins, as described herein.

Also provided herein, in some aspects, are isolated nucleic acid sequences encoding the synthetic proteins and protein ligands, and engineered cells genetically modified with the nucleic acids encoding the synthetic proteins and protein ligands described herein.

Accordingly, in some aspects, provided herein are synthetic inhibitor proteins comprising a Notch NRR (Negative Regulatory Region)-binding scFV fused to a transmembrane domain.

In some aspects, provided herein are synthetic auto-inhibitory proteins comprising a Notch NRR (Negative Regulatory Region)-binding scFV fused to a transmembrane domain.

In some embodiments of these aspects and all such aspects described herein, the Notch NRR comprises a Notch NRR1 of SEQ ID NO: 8.

In some embodiments of these aspects and all such aspects described herein, the Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO: 8 or is a variant of Notch NRR1 of SEQ ID NO: 8.

In other aspects, provided herein are synthetic Notch receptor proteins comprising, in N-terminal to C-terminal order and in covalent linkage, (i) a ligand binding domain (LBD), (ii) a mutated Notch NRR (Negative Regulatory Region), (iii) a transmembrane domain, and (iv) an intracellular domain, wherein the mutated Notch NRR is bound with high-affinity by a synthetic inhibitor protein comprising a mutated Notch NRR-binding scFV fused to a transmembrane domain.

In some embodiments of these aspects and all such aspects described herein, the mutated Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO: 8.

In some aspects, provided herein are synthetic Notch receptor proteins comprising, in N-terminal to C-terminal order, and in covalent linkage, (i) a ligand binding domain (LBD), (ii) a scFV that binds to an at least one Notch NRR (Negative Regulatory Region), (iii) a Notch NRR bound by the scFV, (iv) a transmembrane domain, and (v) an intracellular domain.

In some embodiments of these aspects and all such aspects described herein, the Notch NRR comprises a Notch NRR1 of SEQ ID NO: 8.

In some embodiments of these aspects and all such aspects described herein, the Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO: 8.

In some aspects, provided herein are synthetic, drug-dependent protein comprising a ligand binding domain (LBD), an NS3 protease domain, and a transmembrane domain.

In some embodiments of these aspects and all such aspects described herein, the LBD and transmembrane domain comprise a sequence of human Delta ligand.

In some embodiments of these aspects and all such aspects described herein, the NS3 domain comprises a sequence of SEQ ID NO: 32.

In some embodiments of these aspects and all such aspects described herein, the synthetic, drug-dependent protein further comprises a targeting domain.

In some embodiments of these aspects and all such aspects described herein, the transmembrane domain comprises the human Notch1 transmembrane domain of SEQ ID NO: 13 or a variant thereof.

In some embodiments of these aspects and all such aspects described herein, the scFV comprises, in N-terminal to C-terminal order and in covalent linkage, a VH domain, a linker domain, and a VL domain.

In some embodiments of these aspects and all such aspects described herein, the scFV is selected from any one of SEQ ID NOs: 15-27.

In some embodiments of these aspects and all such aspects described herein, the synthetic protein further comprises a signal sequence N-terminal to the LBD.

Also provided herein, in some aspects, are isolated nucleic acid sequences encoding any of the synthetic proteins described herein.

Also provided herein, in some aspects, are engineered cells comprising isolated nucleic acid sequences encoding any of the synthetic proteins described herein.

In some embodiments of these aspects and all such aspects described herein, the engineered cell is an engineered T cell.

Provided herein, in some aspects, are engineered cells comprising (i) a nucleic acid sequence encoding a synthetic inhibitor protein comprising a Notch NRR (Negative Regulatory Region)-binding scFV fused to a transmembrane domain, and (ii) a nucleic acid sequence encoding a synthetic Notch receptor protein comprising a mutated Notch NRR.

In some embodiments of these aspects and all such aspects described herein, the engineered cell is an engineered T cell.

In some embodiments of these aspects and all such aspects described herein, the nucleic acid sequence encoding the synthetic inhibitor protein, the nucleic acid sequence encoding the synthetic Notch receptor protein, or both are under operable control of a drug-inducible promoter.

In some aspects, provided herein, are synthetic, drug-sensitive transcription factors, comprising: a DNA-binding domain (DB); a transcriptional activation domain (TA); and a HCV NS3 protease domain; wherein the HCV NS3 protease domain is located in between the DB and the TA.

In some embodiments of these aspects and all such aspects described herein, the HCV NS3 protease domain comprises cleavage activity.

In some embodiments of these aspects and all such aspects described herein, the cleavage activity activates the transcription factor.

In some embodiments of these aspects and all such aspects described herein, the DB is Gal4 DB or Cas9 DB.

In some embodiments of these aspects and all such aspects described herein, the DB is reverse tetracycline repressor.

In some embodiments of these aspects and all such aspects described herein, the TA is Gal4 TA, VP64 TA, VP64-p65 TA, or VPR TA.

In some embodiments of these aspects and all such aspects described herein, the synthetic transcription factor further comprises at least one fluorescent or at least one SNAP tag.

In some embodiments of these aspects and all such aspects described herein, the tag is located at a N-terminus or a C-terminus of the transcription factor.

In some embodiments of these aspects and all such aspects described herein, the transcription factor further comprises at least one targeting sequence.

In some embodiments of these aspects and all such aspects described herein, the targeting sequence is a transmembrane domain or a nuclear localization sequence.

In some embodiments of these aspects and all such aspects described herein, the tag is located at a N-terminus or a C-terminus of the transcription factor.

In some embodiments of these aspects and all such aspects described herein, the transcription factor further comprises at least one lipid modification.

In some embodiments of these aspects and all such aspects described herein, the lipid modification is myristoylation or palmitoylation.

In some embodiments of these aspects and all such aspects described herein, the at least one lipid modification is located at an N-terminus or a C-terminus of the transcription factor.

Provided herein in some aspects are isolated nucleic acid sequences encoding any of the synthetic transcription factor described herein.

Provided herein in some aspects are engineered cells comprising the isolated nucleic acid sequences encoding any of the synthetic transcription factor described herein.

Definitions

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

The term “antibody” broadly refers to any immunoglobulin (Ig) molecule and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen) comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which are discussed below, and include but are not limited to a variety of forms, including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab, a F(ab′), a F(ab′)2, a Fv antibody, fragments produced by a Fab expression library, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding fragment thereof, bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference) and/or antigen-binding fragments of any of the above (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference). Antibodies also refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen or target binding sites or “antigen-binding fragments.” The antibody or immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule, as is understood by one of skill in the art. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable domain (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains: CH1, CH2, and CH3. Each light chain is comprised of a light chain variable domain (abbreviated herein LCVR as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well-known to those skilled in the art. The chains are usually linked to one another via disulfide bonds.

The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain, and a CH3 domain, and optionally comprises a CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies

The term “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antigen-binding functions of an antibody can be performed by fragments of a full-length antibody. Such antibody fragment embodiments may also be incorporated in bispecific, dual specific, or multi-specific formats such as a dual variable domain (DVD-Ig) format; specifically binding to two or more different antigens (e.g., Notch receptor and a different antigen molecule). Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature, 341: 544-546; PCT Publication No. WO 90/05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123); Kontermann and Dubel eds., Antibody Engineering, Springer-Verlag, N.Y. (2001), p. 790 (ISBN 3-540-41354-5). In addition, single chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870).

An immunoglobulin constant (C) domain refers to a heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

As used herein, the term “target” refers to a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain which has a binding site can selectively bind. The target can be, for example, an intracellular target (e.g., an intracellular protein target) or a cell surface target (e.g., a membrane protein, a receptor protein). Preferably, a target is a cell surface target, such as a cell surface protein.

As described herein, an “antigen” is a molecule that is bound by a binding site on a polypeptide agent, such as a binding protein, an antibody or antibody fragment, or antigen-binding fragment thereof. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule. In the case of conventional antibodies and fragments thereof, the antibody binding site as defined by the variable loops (L1, L2, L3 and H1, H2, H3) is capable of binding to the antigen. The term “antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.

The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by a binding protein. An epitope may be determined by obtaining an X-ray crystal structure of an antibody:antigen complex and determining which residues on the antigen are within a specified distance of residues on the antibody of interest, wherein the specified distance is, 5 Å or less, e.g., 5 Å, 4 Å, 3 Å, 2 Å, 1 Å or any distance in between. In some embodiments, an “epitope” can be formed on a polypeptide both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An “epitope” includes the unit of structure conventionally bound by an immunoglobulin VH/VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation. The terms “antigenic determinant” and “epitope” can also be used interchangeably herein. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. In some embodiments, an epitope comprises of 8 or more contiguous or non-contiguous amino acid residues in the sequence in which at least 50%, 70% or 85% of the residues are within the specified distance of the antibody or binding protein in the X-ray crystal structure.

The terms “specificity” or “specific for” refers to the number of different types of antigens or antigenic determinants to which a binding protein, antibody or antibody fragment, or antigen-binding portion thereof thereof as described herein can bind. The specificity of a binding protein, antibody or antibody fragment, or antigen-binding portion thereof thereof can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation (KD) of an antigen with an antigen-binding protein, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein, such as a binding protein, antibody or antibody fragment, or antigen-binding portion thereof thereof: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest. Accordingly, a binding protein, antibody or antibody fragment, or antigen-binding portion thereof thereof as defined herein is said to be “specific for” a first target or antigen compared to a second target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a KD value) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to another target or polypeptide.

Accordingly, as used herein, “selectively binds” or “specifically binds” or “specific binding” in reference to the interaction of an antibody, or antibody fragment thereof, or a binding protein described herein, means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope or target) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. In certain embodiments, a binding protein or antibody or antigen-binding fragment thereof that specifically binds to an antigen binds to that antigen with a K_(D) greater than 10⁻⁶ M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, 10⁻¹⁴ M. In other embodiments, a binding protein or antibody or antigen binding fragment thereof that specifically binds to an antigen binds to that antigen with a K_(D) between 10⁻⁶ and 10⁻⁷ M, 10⁻⁶ and 10⁻⁸ M, 10⁻⁶ and 10⁻⁹ M, 10⁻⁶ and 10⁻¹⁰ M, 10⁻⁶ and 10⁻¹¹ M, 10⁻⁶ and 10⁻¹² M, 10⁻⁶ and 10⁻¹³ M, 10⁻⁶ and 10⁻¹⁴ M, 10⁻⁹ and 10⁻¹⁰ M, 10⁻⁹ and 10⁻¹¹ M, 10⁻⁹ and 10⁻¹² M, 10⁻⁹ and 10⁻¹³ M, 10⁻⁹ and 10⁻¹⁴ M. In some embodiments, a binding protein or antibody or antigen-binding fragment thereof binds to an epitope, with a K_(D) 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay. In certain embodiments, a binding protein or antibody or antigen-binding fragment thereof is said to “specifically bind” an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Binding proteins, antibodies or antigen-binding fragments that bind to the same or similar epitopes will likely cross-compete (one prevents the binding or modulating effect of the other). Cross-competition, however, can occur even without epitope overlap, e.g., if epitopes are adjacent in three-dimensional space and/or due to steric hindrance.

The term “antibody fragment,” or “antigen-binding fragment” as used herein, refer to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv) the Fd′ fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)2 fragments, a bivalent fragment including two Fab′ fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) “diabodies” with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

An “Fv” fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F(ab′) 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “monoclonal antibody” or “mAb” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597 (1991), for example. A monoclonal antibody can be of any species, including, but not limited to, mouse, rat, goat, rabbit, and human monoclonal antibodies. Various methods for making monoclonal antibodies specific for an antigen, such as Notch, as described herein, are available in the art. For example, the monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Pat. No. 4,816,567). “Monoclonal antibodies” can also be isolated from or produced using phage antibody libraries using the techniques originally described in Clackson et al., Nature 352:624-628 (1991), Marks et al., J. Mol. Biol. 222:581-597 (1991), McCafferty et al., Nature, 348:552-554 (1990), Marks et al., Bio/Technology, 10:779-783 (1992)), Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993), and techniques known to those of ordinary skill in the art.

The term “human antibody” includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies that comprise heavy and light chain variable domain sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable domains linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies that comprise heavy and light chain variable domain sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable domains in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.

The term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable domains of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable domains. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable domain capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable domain of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2, and L3 or H1, H2, and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan et al. ((1995) FASEB J. 9:133-139) and MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although exemplary embodiments use Kabat or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. ((1987) J. Mol. Biol. 196: 901-917); and Chothia et al. ((1992) J. Mol. Biol. 227: 799-817), both are incorporated herein by reference). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.

As used herein, “antibody variable domain” refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; i.e., CDR1, CDR2, and CDR3), and Framework Regions (FRs). Each heavy chain is composed of a variable region of the heavy chain (VH refers to the variable domain of the heavy chain) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain is composed of a variable region of said light chain (VL refers to the variable domain of the light chain) and a constant region of the light chain. The light chain constant region consists of a CL domain. The VH and VL regions can be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs that are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art. According to the methods used herein, the amino acid positions assigned to CDRs and FRs can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies or antigen binding fragments is also according to that of Kabat.

The term “multivalent binding protein” denotes a binding protein comprising two or more antigen binding sites. A multivalent binding protein may be engineered to have three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets.

Similarly, unless indicated otherwise, the expression “multivalent antibody” is used throughout this specification to denote an antibody comprising three or more antigen binding sites. For example, the multivalent antibody is engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.

In some embodiments, the binding protein is a single chain dual variable domain immunoglobulin protein. The terms “single chain dual variable domain immunoglobulin protein” or “scDVD-Ig protein” or scFvDVD-Ig protein” refer to the antigen binding fragment of a DVD molecule that is analogous to an antibody single chain Fv fragment. scDVD-Ig proteins are described in U.S. Ser. Nos. 61/746,659; 14/141,498 (US application 2014/0243228); and Ser. No. 14/141,500 (US application 2014/0221621), which are incorporated herein by reference in their entireties. In an embodiment, the variable domains of a scDVD-Ig protein are antibody variable domains. In an embodiment, the variable domains are non-immunoglobulin variable domains (e.g., receptor).

In some embodiments, the binding protein is a DVD-Fab. The terms “DVD-Fab” or fDVD-Ig protein” refer to the antigen binding fragment of a DVD-Ig molecule that is analogous to an antibody Fab fragment. fDVD-Ig proteins are described in U.S. Ser. Nos. 61/746,663; 14/141,498 (US Application 2014/0243228); and Ser. No. 14/141,501 (US application US 2014/0235476), incorporated herein by reference in their entireties.

In some embodiments, the binding protein is a receptor DVD-Ig protein. The terms “receptor DVD-Ig protein” constructs, or “rDVD-Ig protein” refer to DVD-Ig constructs comprising at least one receptor-like binding domain. rDVD-Ig proteins are described in U.S. Ser. Nos. 61/746,616; and 14/141,499 (US application 2014/0219913), which are incorporated herein by reference in their entireties.

The term “receptor domain” (RD), or receptor binding domain refers to the portion of a cell surface receptor, cytoplasmic receptor, nuclear receptor, or soluble receptor that functions to bind one or more receptor ligands or signaling molecules (e.g., toxins, hormones, neurotransmitters, cytokines, growth factors, or cell recognition molecules).

The terms multi-specific and multivalent IgG-like molecules or “pDVD-Ig” proteins are capable of binding two or more proteins (e.g., antigens). pDVD-Ig proteins are described in U.S. Ser. No. 14/141,502 (US Application 2014/0213771), incorporated herein by reference in its entirety. In certain embodiments, pDVD-Ig proteins are disclosed which are generated by specifically modifying and adapting several concepts. These concepts include but are not limited to: (1) forming Fc heterodimer using CH3 “knobs-into-holes” design, (2) reducing light chain missing pairing by using CH1/CL cross-over, and (3) pairing two separate half IgG molecules at protein production stage using “reduction then oxidation” approach.

In certain embodiments, a binding protein disclosed herein is a “half-DVD-Ig” comprised of one DVD-Ig heavy chain and one DVD-Ig light chain. The half-DVD-Ig protein preferably does not promote cross-linking observed with naturally occurring antibodies which can result in antigen clustering and undesirable activities. See U.S. Patent Publication No. 2012/0201746 which is incorporated by reference herein in its entirety. In some embodiments, the binding protein is a pDVD-Ig protein. In one embodiment, a pDVD-Ig construct may be created by combining two halves of different DVD-Ig molecules, or a half DVD-Ig protein and half IgG molecule.

In some embodiments, the binding protein is an mDVD-Ig protein. As used herein “monobody DVD-Ig protein” or “mDVD-Ig protein” refers to a class of binding molecules wherein one binding arm has been rendered non-functional. mDVD-Ig proteins are described in U.S. Ser. No. 14/141,503 (US Application 2014/0221622) incorporated herein by reference in its entirety.

The Fc regions of the two polypeptide chains that have a formula of VDH−(X1)n−C−(X2)n may each contain a mutation, wherein the mutations on the two Fc regions enhance heterodimerization of the two polypeptide chains. In one aspect, knobs-into-holes mutations may be introduced into these Fc regions to achieve heterodimerization of the Fc regions. See Atwell et al. (1997) J. Mol. Biol. 270:26-35.

In some embodiments, the binding protein is a cross-over DVD-Ig protein. As used herein “cross-over DVD-Ig” protein or “coDVD-Ig” protein refers to a DVD-Ig protein wherein the cross-over of variable domains is used to resolve the issue of affinity loss in the inner antigen-binding domains of some DVD-Ig molecules. coDVD-Ig proteins are described in U.S. Ser. No. 14/141,504, incorporated herein by reference in its entirety.

The term “bispecific antibody”, as used herein, refers to full-length antibodies that are generated by quadroma technology (see Milstein et al. (1983) Nature 305: 537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz et al. (1985) Nature 314: 628-631), or by knob-into-hole or similar approaches which introduces mutations in the Fc region (see Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14): 6444-6448), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC). By this definition, a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen it binds.

The term “dual-specific antibody”, as used herein, refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see PCT Publication No. WO 02/02773). Accordingly a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

A “functional antigen binding site” of a binding protein is one that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same.

As used herein, the terms “donor” and “donor antibody” refer to an antibody providing one or more CDRs. In an exemplary embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term “donor antibody” refers to a non-human antibody providing one or more CDRs.

As used herein, the terms “acceptor” and “acceptor antibody” refer to the antibody providing or nucleic acid sequence encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term “acceptor” refers to the antibody amino acid providing or nucleic acid sequence encoding the constant region(s). In yet another embodiment, the term “acceptor” refers to the antibody amino acid providing or nucleic acid sequence encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term “acceptor” refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In accordance with this embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well known in the art, antibodies in development, or antibodies commercially available).

As used herein, the term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin. (See, e.g., Shapiro et al. (2002) Crit. Rev. Immunol. 22(3): 183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol. 484:13-30). One of the advantages provided by various embodiments of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

An “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term “linear antibodies” refers to the antibodies described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “humanized antibody” refers to antibodies that comprise heavy and light chain variable domain sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. Accordingly, “humanized” antibodies are a form of a chimeric antibody, that are engineered or designed to comprise minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). As used herein, a “composite human antibody” or “deimmunized antibody” are specific types of engineered or humanized antibodies designed to reduce or eliminate T cell epitopes from the variable domains.

One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Also “humanized antibody” is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′).sub.2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain. A humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype including without limitation IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.

With respect to constructing DVD-Ig or other binding protein molecules, a “linker” is used to denote a single amino acid or a polypeptide (“linker polypeptide”) comprising two or more amino acid residues joined by peptide bonds and used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123).

A “human antibody,” “non-engineered human antibody,” or “fully human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al. Proc. Natl. Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous mouse immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the human antibody can be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes can be recovered from an individual or can have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

An “affinity matured” antibody is one with one or more alterations in one or more CDRs thereof which result an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. A variety of procedures for producing affinity matured antibodies are known in the art. For example, Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992). Selective mutation at selective mutagenesis positions and at contact or hypermutation positions with an activity enhancing amino acid residue is described in U.S. Pat. No. 6,914,128.

A “functional antigen binding site” of an antibody is one which is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same. For multimeric antibodies, the number of functional antigen binding sites can be evaluated using ultracentrifugation analysis as described in Example 2 of U.S. Patent Application Publication No. 20050186208. According to this method of analysis, different ratios of target antigen to multimeric antibody are combined and the average molecular weight of the complexes is calculated assuming differing numbers of functional binding sites. These theoretical values are compared to the actual experimental values obtained in order to evaluate the number of functional binding sites.

As used herein, a “blocking” or “neutralizing” binding protein, antibody, antibody fragment, antigen-binding fragment or an antibody “antagonist” is one which inhibits or reduces the biological activity of the antigen it specifically binds to the antigen. In certain embodiments, blocking or neutralizing antibodies or antagonist antibodies completely inhibit the biological activity of the antigen. The neutralizing binding protein, antibody, antigen-binding fragment thereof can bind a target, such as Notch, and reduce a biological activity by at least about 20%, 40%, 60%, 80%, 85%, or more. Inhibition of a Notch biological activity by a neutralizing binding protein, antibody or antigen-binding fragment thereof can be assessed by measuring one or more indicators of Notch biological activity well known in the art.

An antibody having a “biological characteristic” or “functional characteristic” of a designated antibody is one which possesses one or more of the biological properties of that antibody which distinguish it from other antibodies that bind to the same antigen, including, for example, binding to a particular epitope, an EC50 value, IC50 value or KD values, as defined elsewhere herein.

In order to screen for antibodies which bind to an epitope on an antigen bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed.

As used herein, “antibody mutant” or “antibody variant” refers to an amino acid sequence variant of the species-dependent antibody wherein one or more of the amino acid residues of the species-dependent antibody have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with the species-dependent antibody. In one embodiment, the antibody mutant will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the species-dependent antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side-chain properties, see below) with the species-dependent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence outside of the variable domain shall be construed as affecting sequence identity or similarity.

As used herein, a “targeting sequence” refers to a polypeptide sequence sufficient to direct the localization of, e.g., a polypetide, to a specific subcellular localization. By way of example, a “targeting sequence” can direct the polypeptide to the, e.g., a transmembrane domain, or to the nucleus, e.g., a nuclear localization sequence. A targeting sequence can be added to a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate) to direct the polypeptides localization. A targeting sequence can result in the irreversible or reversible localization of a polypeptide.

An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). See also Jonsson U. et al., (1993) Ann. Biol. Clin., 51:19-26; Jonsson U. et al., (1991) BioTechniques, 11:620-627 (1991); Johnsson U. et al., (1995) J. Mol. Recognit., 8:125-131; and Johnsson U. et al., (1991) Anal. Biochem., 198:268-277.

The term “binding protein conjugate” or “antibody conjugate” refers to a binding protein or antibody or antigen-binding fragment thereof as described herein chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. Preferably the therapeutic or cytotoxic agents include, but are not limited to, anti-cancer therapies as discussed herein, as well as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. When employed in the context of an immunoassay, a binding protein conjugate or antibody conjugate may be a detectably labeled antibody, which is used as the detection antibody.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

The terms “crystal” and “crystallized” as used herein, refer to a binding protein, antibody or antigen-binding protein, or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter that is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as DVD-Igs), or molecular assemblies (e.g., antigen/binding protein complexes).

By “fragment” is meant a portion of a polypeptide, such as a binding protein, antibody or antibody fragment, or antigen-binding portion thereof thereof, or nucleic acid molecule that contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of the reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200 amino acids or more.

By “reduce” or “inhibit” is meant the ability to cause an overall decrease preferably of 10% or greater, 15% or greater 20% or greater, 25% or greater, 30% or greater, 35% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater, or complete or 100% in a parameter, activity, or condition being measured.

The terms “cell lines,” “host cells,” and “host cells lines” refer to cells that can be genetically engineered to express a nucleic acid sequence encoding any of the synthetic proteins or components thereof described herein. Cell lines are typically derived from a lineage arising from a primary culture that can be maintained in culture for an unlimited time. Genetically engineering the cell line involves transfecting, transforming or transducing the cells with a recombinant polynucleotide molecule, and/or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) so as to cause the host cell to express a synthetic protein of interest.

The term “mammalian host cell” is used to refer to a mammalian cell which is capable of being transfected with a nucleic acid sequence and then of expressing a selected recombinant protein of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present. Suitable mammalian cells for use in the present invention include, but are not limited to Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, human HeLa cells, monkey COS-1 cell, human embryonic kidney 293 cells, mouse myeloma NSO and human HKB cells (U.S. Pat. No. 6,136,599). The other cell lines are readily available from the ATCC.

The term “recombinant” when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid, such as a nucleic acid sequence encoding a synthetic Notch protein, i.e., a nucleic acid sequence encoding any such recombinant protein of interest. Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and reintroduced into the cell by artificial means. The term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, using techniques such as Crispr, and related techniques. A “recombinant protein” is one which has been produced by a recombinant cell.

As used herein, the terms “recombinant cell,” “recombinant cell line,” or “modified cell line” refers to a cell line either transiently or stably transformed with one or more nucleic acid constructs, as described herein. Polynucleotides, genetic material, recombinant DNA molecules, expression vectors, and such, used in the compositions and methods described herein can be isolated using standard cloning methods such as those described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; which is incorporated herein by reference). Alternatively, the polynucleotides coding for a recombinant protein product used in the compositions and methods described herein can be synthesized using standard techniques that are well known in the art, such as by synthesis on an automated DNA synthesizer.

Peptides, polypeptides and proteins that are produced by recombinant animal cell lines using the cell culture compositions and methods described herein can be referred to as “recombinant protein of interest,” “recombinant peptide,” “recombinant polypeptide,” and “recombinant protein.” The expressed protein(s) can be produced intracellularly or secreted into the culture medium from which it can be recovered and/or collected. Accordingly, the term “recombinant protein of interest” refers to a protein or fragment thereof expressed from an exogenous nucleic acid sequence introduced into a host cell.

As used herein, the term “transfection” is used to refer to the uptake of an exogenous nucleic acid by a cell, and a cell has been “transfected” when the exogenous nucleic acid has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein.

The term “transformation” as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transfection, the transforming nucleic acid can recombine with that of the cell by physically integrating into a chromosome of the cell, can be maintained transiently as an episomal clement without being replicated, or can replicate independently as a plasmid. A cell is considered to have been stably transformed when the transforming nucleic acid is replicated with the division of the cell.

As used herein an “expression vector” refers to a DNA molecule, or a clone of such a molecule, which has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that would not otherwise exist in nature. DNA constructs can be engineered to include a first DNA segment encoding an acetylation-resistant engineered PDCL3 polypeptide described herein operably linked to additional DNA segments encoding a desired recombinant protein of interest. In addition, an expression vector can comprise additional DNA segments, such as promoters, transcription terminators, enhancers, and other elements. One or more selectable markers can also be included. DNA constructs useful for expressing cloned DNA segments in a variety of prokaryotic and eukaryotic host cells can be prepared from readily available components or purchased from commercial suppliers.

By “cell culture” or “culture” is meant the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, can be used.

As used herein, “cell culture medium” is a media suitable for growth of animal cells, such as mammalian cells, in in vitro cell culture. Cell culture media formulations are well known in the art. Typically, cell culture media are comprised of buffers, salts, carbohydrates, amino acids, vitamins and trace essential elements. “Serum-free” applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum. Various tissue culture media, including defined culture media, are commercially available.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict Notch inhibition by cis-interacting proteins. FIG. 1A shows an illustration of natural cis-inhibition that occurs when Notch and a Notch ligand are expressed on the same cell surface. Interaction of the Notch receptor with its ligand prevents its activation by ligands on adjacent cells. FIG. 1B shows Notch inhibition using a synthetic cis-interacting protein comprising an inhibitory anti-NRR scFv fused to a transmembrane domain. The scFv “clamps” the NRR, preventing its rupture and thus inhibiting Notch activation.

FIGS. 2A-2C show mutating the affinity of the scFv to control force sensitivity. FIG. 2A show a figure depicting the crystal structures of “Ab2” (the scFv used in cis-clamp and as LNR4 domain, left) and the Notch1 NRR (right). Wu et al (2010). Availability of crystal structures allows structure-guided mutations to be made to the scFv that can decrease affinity and tune the stabilizing effects of the scFv. The R99 residue was mutated on the heavy chain of Ab2 to either a lysine (slight reduction in affinity) or an alanine (greater reduction in affinity). FIGS. 2B-2C show co-expression of the Notch receptor with the scFv as a separate transmembrane cis-inhibitor in reporter cells, and cultured them (FIG. 2B) on surface-adsorbed ligand or (FIG. 2C) with ligand-expressing cells. Unaltered Ab2 has inhibitory effects comparable to DAPT (gamma secretase inhibitor), and residue mutations follow expected trends of restoring Notch activity.

FIGS. 3A-3B illustrate an autoinhibitory approach to controlling mechanical sensitivity of Notch. FIG. 3A illustrates a schematic of Notch activation. In the resting autoinhibited conformation (i), the three LNR domains sterically block the S2 cleavage site necessary for Notch activation. Upon application of force (ii), the LNR domains are displaced, allowing the activating proteolytic cleavages (iii) that release the intracellular domain (ICD) to regulate gene expression in the nucleus. FIG. 3B shows the protein structure and schematic of the wild type Notch1 NRR (i), which opens in response to about 5 pN of force. Protein structure of the Notch1 NRR in the presence of an NRR-binding scFv, and schematic of the NRR expressed with this scFv as a contiguous part of its structure (ii). This scFv is designed to act as an “LNR4” domain, which should increase the threshold of force activation to >5 pN.

FIGS. 4A-4C show expression of Notch receptors with and without LNR4 domains in HEK 293FT cells. Cells are made to express Notch-based receptors that comprise: a WT Notch LBD, an NRR1 Notch core with or without LNR4, and a Gal4-VP64 ICD. FIG. 4A shows immunohistochemistry of ligand binding domain (LBD) and ICD showing that cells are able to stably express Notch-based receptors that include an LNR4 domain. LBD stained with soluble ligand D114/Fc; ICD stained with αGal4 antibody. FIG. 4B shows the LNR4 domain successfully binds the NRR. In the WT Notch core, immunostaining the NRR with an NRR-binding antibody revealed co-localization between the LBD and NRR stains. However, the inclusion of an NRR-binding LNR4 domain sterically inhibits the ability to stain the NRR; co-localization between LBD and NRR signals was not observed. LBD stained with soluble ligand D114/Fc; NRR stained with αNRR antibody. FIG. 4C depicts western blot assays in transiently transfected cells that show production of full length receptors at expected masses, with inclusion of an LNR4 domain showing the expected increase in apparent mass (211 vs 239 kDa).

FIGS. 5A-5F show LNR4 increases force threshold of Notch activation in Tension Gauge Tether (TGT) assay. FIG. 5A-5E depict the use of ruptureable double-stranded DNA tethers. Wang et al (2013). One strand of the TGT attaches to a rigid plate through biotin-avidin binding, while the other presents a ligand. The TGT ruptures at a defined force, determined by the orientation of the ligand and the biotin. If the TGT is weaker than the force required to activate the Notch receptor, it ruptures before the LNR domains can be displaced. If it is stronger, the LNR domains are displaced and activation occurs. FIG. 5F shows flow cytometry data of mCherry reporter activity that shows that the WT NRR (which activates at 5 pN) is activated by both 12 pN and 56 pN TGTs. Receptors with an LNR4 domain are activated on 56 pN, but not 12 pN TGTs, indicating decreased mechanical sensitivity.

FIGS. 6A-6C show a schematic depicting drug-dependent Notch activation by the NS-inhibitor BILN-2061 (triangle). FIG. 6A shows that in the absence of drug, the DLL extracellular domain is cleaved from the transmembrane domain (TMD); the resulting soluble DLL ligand is not able to activate Notch receptors. FIG. 6B demonstrates that in the presence of drug, NS3 cleavage is blocked and the DLL extracellular domain remains bound to its TMD in the signaling-competent state. Subsequent binding of the tethered DLL to Notch receptors on an adjacent cell leads to Notch activation and liberation of the Notch intracellular domain (NICD). The liberated NICD is then free to enter the nucleus to mediate gene expression. FIG. 6C shows fluorescence imaging of DLL-NS3-expressing cells and Notch expressing cells (nuclei). Upon activation, Notch-expressing cells exhibit YFP nuclear fluorescence from NICD-mediated activation of a chromosomal histone H2B-YFP gene. In the absence of drug, H2B-YFP is not detected, indicating the absence of Notch signaling. In the presence of drug, however, H2B-YFP expression is observed in Notch-expressing cells adjacent to DLL-NS3-expressing cells, indicating that trans-cellular signaling between these cells is dependent on the inhibition of NS3 activity.

FIGS. 7A-7B depict the structure and activation of Notch receptors. FIG. 7A shows domain topology of the Notch receptor. FIG. 7B shows that Notch is activated upon the binding and endocytosis of ligands presented by neighboring cells. The force applied to the receptor during ligand endocytosis serves to unfold the NRR and initiate successive proteolytic cleavages at S2 and S3. Activation results in the liberation of the NICD, such that it can be transported into the nucleus to effect transcriptional changes.

FIGS. 8A-8D illustrate design and expression of an exemplary hN1-scFv. FIG. 8A shows depictions of an exemplary hN1-scFv with the integrated antibody domain. FIG. 8B shows depictions of hN1-scFv with the reported x-ray structure of the scFv:NRR complex shown in surface rendering. FIG. 8C shows the structure of the scFv:NRR complex with the scFv show as a ribbon diagram{circumflex over ( )} LNRs. FIG. 8D shows fluorescence images of live cells stained with a dye-conjugated version of the Notch ligand DLL4. Membrane staining of hN1-scFv-expressing cells indicate that the chimera is correctly trafficked to the surface.

FIGS. 9A-9B show hN1-scFv expression requires increased force for activation. FIG. 9A shows hN1-scFv-expressing Notch reporter cells that contain a NICD-dependent histone-YFP are not activated when co-incubated cells expressing the Notch ligand DLL1. FIG. 9B shows plating of the same cells on DLL1-coated coverslips are able to activate hN1-scFv, due to the ligand ability to apply increased tension to the receptor when bound to a stiff substrate.

FIG. 10 shows that recombinant hN1 is predominantly localized to the ER in CHO lines. Expression of anti-NRR-scFv-TMD increases the level of hN1 localized to the cell surface.

FIGS. 11A-11D show drug dependent localization and activity of dCNV. FIG. 11A is an illustration depicting the self-immolation of dCNV. cis-cleavage leads to the separation of dCas9 from its fused nuclear localization (NLS) and transactivation (VPR) domains. FIG. 11B shows that dCNV is stabilized upon exposure to the NS inhibitor ciluprevir. Immunoblot using anti-Cas9 shows complete cleavage of dCNV in the absence of drug, producing a 160 kDa band (*) corresponding to the cleaved dCas9. In drug treated cells, full-length (**, 260 kDa) dCNV is observed. FIG. 11C shows images of immunostained cells showing drug-dependent localization of dCas9, which localizes to the in drug-treated cells due to preservation of the fused NLS. FIG. 11D shows that dCNV in combination with an sgRNA targeting the promoter region CXCR4 is used to upregulate the expression of the chemokine receptor in a drug-dependent manner. Relative expression levels were quantified by flow cytometry using an anti-CXCR4.

FIG. 12 demonstrates inducible inhibition of Notch signaling with a Tet-operated scFV. Cells expressing Notch-Gal4 alone (top three peaks), with a constitutive scFv (middle three peaks), or with a Tet-operated scFv (bottom three peaks) are cultured with sender cells expressing no ligand (ctrl) or the ligand Dll1. The constitutively expressed scFv inhibits Notch activation in the presence of ligand, while the Tet-operated scFv only inhibits activation in the presence of drug (doxycycline).

FIGS. 13A-13C demonstrate a strategy for designing synthetic NRR-scFv pairs. FIG. 13A shows a figure describing the specificity of scFv's against NRR1 and NRR2 to each NRR and various chimeras of the two. Wu et al (2010). Chimeras that bind neither scFv are promising starting points for choosing a synthetic NRR domain whose scFv may be orthogonal to each WT NRR. FIG. 13B shows sequence alignments of NRR1 and NRR2. FIG. 13C shows sequence alignments of scFv's against NRR1 and NRR2. FIGS. 13B and 13C are paired with the NRR-scFv crystal structure (FIG. 2A). This is valuable for understanding the regions of each scFv that bind portions of each NRR and subsequently designing chimeric scFv's.

FIG. 14 shows an exemplary drug-dependent NS3-Gal4 transcription factor. NS3 fused between the BD and AD of Gal4 yields a functional transcription factor. This construct is intact and active in the presence of an NS3-inhibiting drug, but cleaved and inactive in the absence of drug.

FIGS. 15A-15I shows drug inducible “turn-on” and “turn-off” transcription factors (TFs). FIG. 15A Schematic depicting a general exemplary “turn-on” TF design and its stabilization in the presence of an NS3 inhibitor. FIG. 15B Western blot showing the preservation of full-length DBGal4-NS3-TAGal4 (anti-HA; 60.6 kDa) in cells treated with increasing concentrations of BILN-2061. TF stabilization was accompanied by a corresponding increase in the expression of the H2B-Citrine reporter protein (46.5 kDa). DBGal4-NS3-TAGal4 was detected using an HRP-conjugated anti-HA antibody, and signal enhancement was achieved through application of an HRP-conjugated secondary antibody Enhanced detection showed TF stabilization in cells treated with 0.08 μM of BILN-2061; intact TF remained undetectable in drug-untreated controls. An additional band corresponding to Cho-K1 mediated degradation of the fusion construct was also detected. FIG. 15C Fluorescence images of a Cho-K1 reporter cells (UAS-H2B-Citrine) stably expressing DBGal4-NS3-TAGal4 in the absence and presence of 2.5 μM BILN-2061. FIG. 15D Comparison of drug-induced reporter expression levels (as measured by flow cytometry) of cells expressing “turn-on” TFs containing the indicated TA domains. FIG. 15E Schematic of TMD-NS3-Gal4min cleavage and its drug-activated “turn-off” activity. General design of the “turn-off” TFs containing a generic membrane-localizing element FIG. 15F Fluorescence images of HEK 293A cells expressing the dual-tagged BFP-TMD-NS3-Gal4-mCherry. The mCherry-tagged TF unit localized to the nucleus in the absence of drug and was targeted to ER and PM in cells treated with 3 μM BILN-2061. BFP is N-terminal to the TMD; Gal4 is tagged with mCherry. Scale bar is 10 μm. FIG. 15G Schematic of myr-palm-NS3-Gal4min cleavage and stabilization. FIG. 15H Immunostained HeLa cells showing the sequestration and membrane-targeting of Gal4_(min) in drug-treated cells expressing myr-palm-NS3-Gal4min. FIG. 15I Flow cytometry analysis of transiently transfected HEK 293FT cells coexpressing DBrTetR-NS3-TAVP64-p65 and TMD-NS3-Gal4_(m)i_(n) and containing TRE BFP and UAS H2B-Citrine reporter constructs. Treatment of cells with increasing concentration of BILN-2061 induced concurrent TRE BFP activation and UAS H2B-Citrine repression. Analyses were carried out in media containing 100 ng/mL doxycycline to induce DBr_(TetR) binding to tetO-DNA.

FIGS. 16A-16E show drug-control over endogenous gene expression using dCas9-NS3-NLS/VPR. FIG. 16A Schematic depiction of the drug-inducible stabilization and nuclear localization of dCas9-NS3-NLS/VPR. FIG. 16B Western blot showing the preservation of full-length dCas9-NS3-NLS/VPR copies (250 kDa) in cells treated with 3 μM BILN-2061. An unfused dCas9 domain (160 kDa) is produced in the absence of drug. FIG. 16C Immunostained HeLa cells showing the subcellular localization of the dCas9 domain in the presence or absence of 3 μM BILN-2061. Nuclear localized dCas9 was observed only in drug treated cells. FIG. 16D Schematic of the time-dependent dye labeling strategy used to analyze cells expressing SNAP-dCas9-NS3-NLS/VPR (top), and corresponding fluorescence images of dye-labeled HeLa cells (bottom). Old protein copies produced in the absence of drug were confined to the cytoplasm, whereas those that were made in the presence of drug were able to translocate into the nucleus. FIG. 16E Drug-induced upregulation of CXCR4 expression in HEK 293FT cells coexpressing dCas9-NS3-NLS/VPR and sgRNA targeting the endogenous CXCR4 promoter. Expression of the chemokine receptor was analyzed by staining of cells anti-CXCR4 antibody followed by quantification via flow cytometry.

FIGS. 17A-17F show drug control over ligand presentation and intercellular Notch signaling FIG. 17A Schematic of representation of an exemplary design of Dll1-NS3-mCherry and FIG. 17B shows its drug-inducible preservation as a cell-surface ligand. FIG. 17C Immunofluorescence staining of fixed (non-permeabilized) cells expressing Dll1-NS3-mCherry. Surface presentation of the Dll1 extracellular region was induced by treatment of cells with 1.5 μM BILN-2061. FIG. 17D Schematic depiction of trans-cellular signaling between Notch “receiver” cells by Dll1-NS3-mCherry expressing “sender” cells in the absence and presence of drug. FIG. 17E Fluorescence images of cocultured sender and receiver cells. Sender cells expressed the Dll1-NS3-mCherry ligand. Receiver cells constitutively expressed human Notch1 and H2B-Cerulean, and conditionally expressed H2B-Citrine upon up Notch activation. Activated receiver cells were observed only in cocultures treated with drug (1.5 μM BILN-2061). The interface between sender and receiver cells colonies is denoted by the white dotted line. FIG. 17F Magnified region from the inset shown in (17E) shown as an overlay with the corresponding transmitted light image.

FIG. 18 show comparison various commercially available NS3 inhibitors. A clonal Cho-K1 derived cell line containing a stably integrated Gal4-dependent reporter gene (UAS H2B-Citrine) and constitutively expressing DBGal4-NS3-TAGal was tested against various NS3 inhibitors. Cells were treated with drug for ˜24 hours and expression of the H2B-Citrine reporter protein was subsequently quantified by flow cytometry.

FIGS. 19A-19B show characterization of the DBrTetR-NS3-TAVP64-p65 “turn-on” TF. FIG. 19A The presence of both doxycycline (to induce rTetR binding to tetO sequences) and an NS3 inhibitor (BILN-2061) was required to activate the expression of an BFP reporter protein under control of the TRE promoter. FIG. 19B Comparison of BFP expression (as quantified by flow cytometry) in HEK 293FT cells co-transfected with an DBrTetR-NS3-TAVP64-p65-encoding plasmid and reporter TRE-BFP DNA and treated with the indicated drugs.

FIGS. 20A-20B show drug-induced gene downregulation using “turn-off” TFs. FIG. 20A Western blot analysis of HEK 293FT cells transiently transfected with DNA encoding TMD-NS3-Gal4min or myr-palm-NS3-Gal4min. Intact proteins were preserved in cells treated with 3 μM BILN-2061. The HEK 293FT cells contained a stably integrated Gal4 dependent reporter gene (UAS H2B-Citrine), the expression of which was detected only in drug untreated cells. The full-length mass of TMD-NS3-Gal4_(min) is 96 kDa, and full-length myr-palm-NS3-Gal4min is 63 kDa. A positive control was carried out in which cells were transfected with a plasmid encoding the Gal4 DB fused to VP64 (Gal4-VP64). FIG. 20B Drug-induced downregulation of reporter expression in HEK 293FT reporter cells (UAS H2B-Citrine) transiently transfected with constructs encoding TMD-NS3-Gal4min or myr-palm-NS3-Gal4_(min) and treated with the indicated concentrations of the NS3 inhibitor grazoprevir. Values are displayed as mean±s.d., as determined in triplicate.

FIGS. 21A-21B show drug-induced activation of a fluorescent reporter gene using dCas9-NS3-NLS/VPR. Flow cytometry FIG. 21A and representative fluorescence images FIG. 21B of H2B-Citrine expression from a reporter construct (UAS H2B-Citrine) as mediated by drug-stabilized dCas9-NS3-NLS/VPR and a corresponding UAS-targeting sgRNA. Flow cytometry analyses of reporter expression levels were compared to activation as mediated by “dCNV S139A,” a version of dCas9-NS3-NLS/VPR in which the catalytic serine residue of the NS3 protease was mutated to alanine. The sgRNA construct used (Addgene plasmid #6415) also encoded a constitutively expressed mCherry.

FIG. 22 sgRNAs targeting the CXCR4 promoter were co-transfected into HEK 293FT alongside DNAs encoding either dCas9-VPR, or dCas9-NS3-NLS/VPR. The extent of CXCR4 upregulation was subsequently quantified by flow cytometry using a fluorescently-labeled anti-CXCR4 antibody. Cells containing dCas9-NS3-NLS/VPR were analyzed under drug-treated and untreated conditions (BILN-2061, 3 μM), and compared to catalytically inactive dCas9-NS3-NLS/VPR (NS3 “S139A” mutant), dCas9-VPR containing, and non-transfected HEK 293FT cells (control). Antibody staining of live cells was carried out 24 hours following transfection/drug-treatment. Values are displayed as mean±s.d., as determined in triplicate.

FIGS. 23A-23B show dose-dependent nuclear localization of the dCas9 domain in cells expressing dCas9-NS3-NLS/VPR. FIG. 23A Transfected HeLa cells were treated with the indicated concentration of BILN-2061 for 24 h and subsequently fixed and permeabilized before immunostaining with anti-Cas9 antibody and counterstaining with DAPI. FIG. 23B The extent of nuclear localization was analyzed through through analysis of the pixel intensities along the lines indicated in (A) and plotted using the ImageJ-based software package Fiji.

FIGS. 24A-24B show inducible gene activation using MCP-NS3-VP64. FIG. 24A Schematic of inducible dCas9-mediated transcription via conditional preservation of a sgRNA-binding protein (MCP-NS3-VP64). MCP binds hairpin-modified sgRNA and localizes the VP64 TA domain to the DB scaffold only in drug-treated cells. FIG. 24B Drug-induced activation of a TRE-H2B-Citrine reporter protein via expression of dCas9 combined with MCP-NS3-VP64 and sgRNA targeting the TRE3G promoter. Cells were co-transfected with fluorescent marker (mTurqoise-2) to identify positively transfected cells. H2B-Citrine was induced only in drug-treated cells.

FIGS. 25A and 25B show images of Dll1-NS3/4A-mCherry mediated cell-cell signaling captured by epifluorescence imaging under 10× magnification. FIG. 25A Sender cells expressing the Dll1-NS3/4A ligand are cultured with receiver cells expressing the Notch transmembrane receptor. Receiver cell population is identified by a constitutively expressed H2B-Cerulean. Notch activation drives reporter activity of H2B-Citrine. Cells are cultured together with or without 1.5 μM BILN-2061 drug for 72 hours before imaging. FIG. 25B

FIGS. 26A-26C show data depicting the flow cytometry gating procedures used herein. FIG. 26A Live cells were gated using FSC and SSC as depicted with the black line. FIG. 26B A positive transfection gate was made by gating for the top 1% fluorescing WT cells. FIG. 26C The positive transfection gate was then applied to all transfected cell populations. Geometric mean of reporter fluorescence was then measured from positively transfected cells. Mean intensities from nuclear H2B-Citrine in individual receiver cells was quantified via analysis of images from drug-treated and untreated co-cultures. Expression of the NICD-dependent reporter was compared between receiver cells that were in direct contact with sender cells, as well those that were distant from sender cells. The displayed values are reported as mean intensities, ±s.e.m., with n>100 cells per analyzed group.

FIGS. 27A and 27B show time-dependent analysis of the drug-induced preservation of Gal4-based TFs containing NS3 upon treatment of cells with grazoprevir. Stable cell lines expressing either Gal4DB-NS3-Gal4TA or Gal4DB-NS3-VP64 were treated with 5 μM grazoprevir for the indicated times prior to cell lysis in SDS-PAGE loading buffer and subsequent analysis by western blot. The drug-induced preservation of intact TF copies was determined via the detection of bands corresponding to the intact masses of each TF. Western blots showing the preservation of full-length copies of FIG. 27A Gal4DB-NS3-Gal4TA (60.6 kDa), and FIG. 27B Gal4DB-NS3-VP64 (57.8 kDa) are displayed. Times refer to the number of minutes in which cells were exposed to drug prior to lysis. Western detection of the TFs was achieved via an HRP-conjugated anti-HA primary antibody, followed by an HRP-conjugated secondary antibody.

FIGS. 28A and 28B show reversibility of drug-induced “turn-on” TF preservation. FIG. 28A HEK 293FT cells transfected with DNA encoding rTetR-NS3-VP64-p65 were “pulsed” with drug (5 μM BILN-2061, 24 h) and subsequently “chased” with drug-free media for the indicated times. Following each chase, cells were lysed in SDS PAGE loading buffer and the lysates were subsequently analyzed via western. Intact rTetR-NS3-VP64-p65 (93.3 kDa) and the VP64-p65 cleavage product (42.0 kDa) were detected via fused HA tag. NT refers to a non-transfected control. FIG. 28B The reversibility of transcriptional activation by Gal4DB-NS3-VP64 was analyzed using a luciferase-based reporter assay. Applying BILN-2061 and grazoprevir as inducers, a pulse-chase analysis was carried out in which cells were treated drug, then withdrawn from drug for chase periods of the indicated times. At the end of the time course, the luciferase activity of cells was quantified using a luminescence assay. The data were obtained using Cho-K1 reporter cells (UAS-H2B-Citrine) containing a stably-integrated Gal4DB-NS3-VP64 construct. The cells were transfected with a Gal4-dependent luciferase reporter construct (5×GAL4-TATA-luciferase) and treated with 3 μM BILN-2061 or grazoprevir 16 h later. The first chase was initiated at the 12 h time point after drug addiction. “Last 12” refers to control cells that were transfected and maintained in drug-free media, and treated for only the last 12 hours preceding cell lysis. Signal from the “Last 12” samples confirmed that the diminished luciferase activity measured in the chased cells was not due to their decreased drug exposure durations. Luminescence values were normalized to signal from a co-transfected NanoLuciferase control construct (pNL1.1.TK[Nluc/TK]). Values are displayed as mean±s.d., as determined in triplicate.

FIGS. 29A and 29B show time-dependent western analysis tracking the degradation of cleaved Gal4_(min) domains. HEK 293FT cells transfected with DNAs encoding either TMD-NS3-Gal4_(min) or TMD-NS3-Gal4_(min)-PEST were grown without inhibitor until treatment with 3 μM grazoprevir at the indicated times prior to lysis in SDS PAGE loading buffer and subsequent analysis by western blot. Detection of the intact and cleaved states of each protein was achieved via labeling with an anti-Gal4 DB antibody on western blots loaded with lysates from cells expressing FIG. 29A TMD-NS3-Gal4_(min), or FIG. 29B TMD-NS3-Gal4_(min)-PEST. Bands corresponding to the full-length version of each construct (“Full Construct”) were detected only in lanes loaded with lysates from drug-treated cells. The intensity of the “Full Construct” bands grew over time, indicating accumulation of the intact proteins following NS3 inhibition. Bands corresponding to cleaved Gal4min and Gal4min-PEST were also observed (“Cleaved TF”), the intensities of which diminished over time. The half-life of the Gal4min-PEST was attenuated relative to that of Gal4_(min). The PEST domain used was derived from the C-terminal region of mouse ornithine decarboxylase, which has previously been used to generate a “destabilized” version of GFP with a reduced half-life of 2 hours.

FIG. 30 shows results using a novel mechanoreceptor with a fluorescent protein as its bulky ectodomain. Green fluorescent protein has been shown to unfold at approximately 100 pN (Dietz 2004), and unfolding of this domain would reduce steric hindrance and in turn allow the release of an intracellular transcription factor through gamma secretase processing. Cells transiently transfected with DNA encoding this receptor display increased activation when plated on wells coated with an antibody that binds it. However, this increase in activation only occurred when the antibodies are tethered, and thus are able to apply force to the receptors: soluble antibodies at similar concentrations do not increase activation. Additionally, this process is supported to be gamma secretase dependent, as addition of a gamma secretase inhibitor diminished cell activation. p=0.000269805 (t-test between coated anti-myc and noncoated for percent cell activation); p=0.000763826 (t-test between coated anti-myc and noncoated for fold above receptor).

FIGS. 31A-31C show reduction of SynNotch leakiness by incorporation of the juxtamembrane LWF motif (FIG. 31A) Schematic of the Notch TMD and LWF motif, slightly modified from [1] to highlight the difference between the original SynNotch core's C-terminus and the modified SynNotch core's C-terminus, where 10 amino acids are added to include the LWF motif (FIGS. 31B and 31C) anti-FITC SynNotch receptors containing SN or SN-LWF as the core domain are expressed and tested in HEK 293FT cells. Cells are either plated on fibronectin alone to test background activation (dashed lines) or on fibronectin with BSA-FITC to activate the receptors (solid lines). FIG. 31A discloses SEQ ID NO: 70. In (FIG. 31B), receptors have the Gal4-VP64 ICD, while in (FIG. 31C), receptors have the more potent Gal4-VPR ICD. Values shown are the percentage of cells in the ON state for each case.

FIGS. 32A and 32B show expression of sNRR-containing receptors. (FIG. 32A) Immunostaining of non-permeabilized HeLa cells expressing GFP-binding SynNotch receptors that contain either the WT NRR (top) or the engineered sNRR domain (bottom). Labeling of a myc tag at the N-terminus of the receptors shows that the sNRR domain does not inhibit the cell's ability to express the receptor at its surface. Next, using a soluble antibody to stain the NRR, it was found that ECD and NRR stains co-localize for the NRR-based receptor, as expected since both protein regions are available at the cell surface. The sNRR domain, however, does not stain for the NRR. This indicates that the incorporated scFv successfully interacts with the NRR domain as intended. (FIG. 32B) Western blot analysis of the cells shown in (A). For both receptors, an αMyc blot is able to detect the full-length (FL) and S1-processed (N-term) forms of the receptor. S1-processing by furin protease cleaves the receptor into a noncovalently joined heterodimer, a feature important for surface expression and activity of Notch receptors.

FIGS. 33A and 33B show increased mechanical strength of sNRR domain in TGT assay. (FIG. 33A) TGTs are double stranded-DNA tethers that rupture at defined forces. One strand of the TGT attaches to a rigid plate, and the other presents a ligand for cells to bind. In this case, the ligand used is fluorescein, which interacts with an αFITC SynNotch. If the tension tolerance of the TGT is weaker than that of the NRR, the TGT will break before the NRR can open. If it is stronger, the NRR domain will unravel, and Notch activation will occur, as visualized by expression of an H2B-mCherry reporter gene. (FIG. 33B) Flow cytometry data and fluorescence imaging of HEK 293FT cells expressing NRR- (left) and sNRR-based (right) SynNotch stimulated with various strengths of TGT ligands. NRR-based receptors activate in response to TGTs 12 pN and stronger, as expected. sNRR-based receptors do not activate until 56 pN of tension tolerance is provided. It is worthwhile to re-emphasize that for the 12, 56, and 100 pN stimuli above, sNRR recepetors are binding the same ligand in each instance, but are able to respond different based off the underlying mechanical properties of the ligand.

FIGS. 34A-34E show tunability of sNRR mechanical strength. (FIG. 34A) Crystal structure (PDB 3L95) of the soluble antibody used to design sNRR in complex with its NRR antigen. (FIG. 34B) Flow cytometry data from a collection of mutated sNRR domains stimulated vs TGTs. For each receptor, values are normalized to their reporter fluorescence on the maximum strength stimulus (>100 pN). (FIG. 34C) Selected flow cytometry data from (FIG. 34B) presented in detail. The original sNRR receptor is insensitive to a 12 pN stimulus, and furthermore can discriminate between 43, 54, and >100 pN. Mutating a Tyr residue to a Phe (Y49F) slightly weakens the receptor, inducing a marginal response to 12 pN and decreased discrimination between 43 pN and above. Further mutating to an Ala (Y49A) causes ˜50% activation in response to 12 pN and abolishes the ability to discriminate between 43 pN and above. The WT NRR is the weakest, responding identically to all mechanical stimuli. Color legend as in (FIG. 34B). (FIG. 34D) Single-cell traces from timelapse imaging of HeLa cells expressing model sNRR domains. This data further confirms the distinct tensile strengths of engineered sNRR domains and reveals their ability to discriminate forces over time. Values are plotted mean±SD, normalized to each receptor's mean max activation in response to >100 pN. Color legend as in (FIG. 34B). (FIG. 34E) Stochastic modeling of sNRR-TGT interactions. A given sNRR-TGT pair is modeled as having a relative probability of either the TGT rupturing of the receptor activating, as determined by the relative mechanical strengths of the two components. Mechanical strengths are modeled as thermal dissociation rates, and four such strengths are considered for sNRR's and TGT's each, similar to the data presented in (FIG. 34D). Values plotted are the number of receptors that get activated during the stochastic model, normalized as in (FIG. 34D), with 10 runs of the model plotted per pair.

FIG. 35 show force-based gene circuits: myogenic differentiation. αFITC SynNotch receptors with an ICD that drives expression of MyoD are expressed in C3H 10T1/2 fibroblasts and stimulated with 12 and 54 pN TGTs. Fibroblast differentiation down a myogenic lineage is identified by the presence of myosin heavy chain and multinucleation. While cells with NRR-based receptors differentiated in response to both stimuli, cells with sNRR-based receptors only differentiate on 54 pN TGTs.

FIG. 36 shows screening of NRR-binding scFv's. Various antibodies known to bind the NRR and inhibit Notch activation are incorporated into SynNotch receptors and stimulated with 12 and 56 pN TGTs. scFv's WC629 and WC75 do not offer detectable mechanical stability, as the receptors are unable to discriminate between the two stimuli, similar to the WT NRR. scFv E6 offers marginal increased mechanostability, while the original sNRR domain offers the greatest mechanical stability, with 56 pN only beginning to stimulate the receptor.

FIG. 37 shows SEQ ID NO: 56, which is a nucleotide sequence encoding TMD-NS3-Gal4_(min). Signal Sequence in double underlined/italics, scfv linker is zigzag underlined, TMD is dotted underlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, DB_(Gal4) in bold, and TA_(Gal4) in bold/dashed underlined.

FIG. 38 shows SEQ ID NO: 57, which is a nucleotide sequence encoding BFP-TMD-NS3-Gal4_(min)-mCherry. Signal Sequence in double underlined/italics, BFP in bold/dashed underlined, TMD in zigzag underlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, DB_(Gal4) in bold, mCherry in italic/dotted underlined, and TA_(Gal4) is bold/dotted-dashed underlined.

FIG. 39 shows SEQ ID NO: 59, which is a nucleotide sequence encoding TMD-NS3-DB_(Gal4)-TA_(VP64). Signal Sequence in italics/double underlined, scfv linker in bold/double underlined, TMD in zigzag underlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, DB_(Gal4) in bold, VP64 is dotted underlined.

FIG. 40 shows SEQ ID NO: 60, which is a nucleotide sequence encoding dCas9-NS3-NLS-VPR. dCas9 in bold/italics/underlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, NLS in bold, VP64 is double underlined, P65 in double underlined/italics, Rta is bold/dotted underlined.

FIG. 41 shows SEQ ID NO: 61, which is a nucleotide sequence encoding dCas9-NS3-NLS-VPR. SNAPf is zigzag underlined, dCas9 in bold/italics/underlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, NLS in bold, VP64 is double underlined, P65 in double underlined/italics, Rta is bold/dotted underlined.

FIG. 42 shows SEQ ID NO: 68, which is an amino acid sequence encoding myc-moxGFP-mN1TMD-GAL4-VP64Bolded text indicates the CD8alpha signal sequence; Bolded, underlined text indicates the myc epitope; Italicized text indicates moxGFP (e.g., as described in Costantini, et al. 2015); Double underlined text indicates the mouse Notch1 juxtamembrane and transmembrane domains; and Zig-zag underlined text indicates GAL4-VP64 Activator.

DETAILED DESCRIPTION

As described herein, receptors with increased or decreased force activation thresholds are both novel and have wide-ranging applications, including, but not limited to, generation of cells with ability to detect physical features of solid tumors, mechanical properties of biomaterials, etc. Furthermore, the cis-clamps described herein permit regulation of engineered cells expressing synthetic Notch receptors or “SynNotch receptors,” such as, for example, therapeutic T cells, and are useful for reducing the known background activity of these receptors. In addition, drug-inducible Notch activation described herein allows tighter control of therapeutic interventions that utilize Notch receptor transduction mechanisms.

A continuing goal of synthetic biology is to be able to program new functions into cells in ways that can be precisely manipulated for applications in medicine and basic research. Toward this end, researchers have recombined modular domains from natural signaling proteins and genetic control elements to produce new biological “parts” for cellular engineering applications.¹ However, a limitation has been the relatively small number of protein components that are available for designing drug-sensitive systems. In particular, domains that can be used to engineer chemical-control into diverse proteins—in ways that allow them to be tightly and selectively regulated using orthogonal drugs—remain lacking.

Accordingly, provided herein, in some aspects, are synthetic Notch receptors or “SynNotch receptors,” having defined and/or programmable force-activation thresholds for applications in cell engineering, T cell immunotherapy, and tissue engineering.

Provided herein, in some aspects, are compositions and methods for regulating SynNotch receptor proteins and reducing their background levels of activity. For example, in some embodiments, genetic regulation of cis-clamps can be used to “turn off” the cell-killing activity of immune cells.

Provided herein, in some aspects, are compositions and methods for drug-inducible control of Notch and/or synNotch activation. Such compositions and methods comprise one or more synthetic proteins, such as a synthetic, drug-dependent protein or a synthetic inhibitor protein. As used herein, a “synthetic protein” refers to a non-naturally occurring protein or polypeptide having a desired function for use in the compositions and methods described herein. Such synthetic proteins can comprise one or more domains from or derived from a naturally occurring protein in combination with one or more domains from or derived from another naturally occurring protein to create a synthetic protein having desirable functions that are not found together naturally. Such domains include naturally occurring domains, as well as mutated or engineered domains derived from naturally occurring domains, or portions of a naturally occurring domain having a desired activity. For example, in some embodiments, a synthetic protein comprises one or more NS3 protease domains or one or more Notch Regulatory Region (NRR)-binding domains. Other examples of domains that can be used in the synthetic proteins described herein include transcriptional activation domains, transcriptional repressor domains, DNA-binding domains, such as zinc-finger-binding domains, protease domains, and the like. Other domains contemplated for use in the synthetic proteins described herein include extracellular domains, such as ligand-binding extracellular domains, transmembrane domains, and intracellular domains, such as intracellular signaling domains. In addition, the nucleic acid sequences encoding the synthetic proteins described herein can comprise additional sequence elements such as signal sequences and tag sequences.

Accordingly, provided herein, in some aspects, are “cis-clamps” or synthetic inhibitor proteins. As used herein, a “synthetic inhibitor protein” comprises a Notch Regulatory Region (NRR)-binding scFv fused to a transmembrane domain and acts to inhibit Notch receptor or synthetic Notch receptor activity. Non-limiting examples of “synthetic inhibitor proteins” described herein include SEQ ID NOs: 42, 42, and 44 and variants thereof having similar or enhanced inhibitory activity. Notch is typically activated by ligands expressed on adjacent cells, but inhibited when ligands are expressed on the same cell through a mechanism known as “cis-inhibition” (FIG. 1A). This cis-interaction serves to prevent cells from receiving signals from their neighbors, and also prevents spontaneous “ligand independent” background activation, reducing Notch background activity. As demonstrated herein, membrane-tethered anti-NRR scFvs can be used as genetically encoded Notch inhibitors, or “cis-clamps” (FIG. 1B). These scFvs are derived from antibodies that bind and stabilize the NRR region of Notch receptors, preventing their activation. As shown herein, these “cis-clamps” or synthetic inhibitor proteins can be used to regulate both endogenous Notch and synthetic Notch (“SynNotch”) activity in a manner similar to ligand cis-inhibition.

As known by those of skill in the art, “single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody as a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

In some embodiments of the aspects described herein, cis-clamps or cis-inhibitors are used for cell engineering applications, such as for use in therapeutic T cells. Notch and/or SynNotch receptors are known to exhibit background “leaky” activation. In engineered cells, these chimeras are used, in some embodiments, as regulatory elements to limit signaling from synthetic Notch receptors and, in some embodiments, to reduce off-target T cell killing in the case of engineered SynNotch T cells.

The cis-clamps described herein are especially useful in situations where ligand co-expression is problematic. For example, typically cis-inhibition of a SynNotch receptor is achieved by co-expressing the receptor and its target ligand on the same cell. However, in the case of cell immunotherapy, ligands used to activate SynNotch receptors on T cells are usually cell surface cancer markers, such that co-expression of these markers would in principle permit cis-inhibition of SynNotch proteins, as well as causing the therapeutic cells to attack one another. Thus, the cis-clamps described herein provide a route through which SynNotch receptors can be regulated without the introduction of cancer marker/antigens to the engineered cells.

In some embodiments, tuning the affinity of an scFv is performed to engineer mechanical sensitivity, as shown in FIGS. 2A-2C, where an NRR-binding scFv is mutated and expressed as a separate transmembrane cis-clamp. Non-limiting examples of NRR-binding scFv sequences that can be used or be further engineered or modified to be used in some embodiments of the synthetic inhibitor proteins described herein include SEQ ID NOs: 15-27.

In those embodiments where amino acid sequence modification(s) of an scFv, such as an scFv of any one of SEQ ID NO: 15-27, is performed to engineer mechanical sensitivity, amino acid sequence variants of the NRR-binding scFv are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the scFv, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the scFv or antibody from which it is derived. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also can alter post-translational processes of the scFv, such as changing the number or position of glycosylation sites.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions for antibody-based sequences include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of an antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis typically are the hypervariable regions of the VH and/or VL domains of the scFv.

Substantial modifications in the biological properties of an scFv can be accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Any cysteine residue not involved in maintaining the proper conformation of antibodies or antibody fragments thereof can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display.

Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition of glycosylation sites to the antibodies or antibody fragments thereof described herein is accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of the scFVs used herein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.

Provided herein, in some aspects, are synthetic Notch receptors with mutated or engineered NRR domains. Mutating the NRR domain and utilizing an scFv that has high affinity to the mutated NRR (but not the native NRR) in either the cis-clamp or auto-inhibitory receptor configurations allow for a more specific system with reduced off-target effects, such as, e.g., the scFv binding the NRR region on notch receptors of adjacent cells).

Also provided herein, in some aspects, are synthetic auto-inhibitory Notch receptors that comprise an NRR-binding scFv portion. Similar to the aspects directed to cis-clamps, the scFv portion in the auto-inhibitory Notch receptor stabilizes the NRR region, resulting in more force being required to activate the receptor. In some embodiments, NRR-binding scFv portions are expressed as a contiguous part of Notch-based synthetic receptors to act as a synthetic “fourth LNR domain” (LNR4), offering additional stability to the NRR and increasing the force threshold of Notch activation. This exemplary approach is described in FIGS. 3A-3B.

In some embodiments, the LNR4 domain can be mutated to change the affinity of binding to the NRR in order to tune the mechanical sensitivity of the receptor, as shown in FIGS. 2A-2C for the cis-clamp embodiment.

In some aspects, provided herein are constructs, compositions, and methods for controlling the binding/activation of endogenous and/or synthetic Notch receptors through the use of synthetic proteins acting as ligands and comprising viral protease domains, such as NS3 protease domains from the hepatitis C virus (HCV). The NS3 domain is a serine protease embedded within the HCV polyprotein that excises itself from the precursor polypeptide by cleaving recognition sites flanking it at either end. The enzyme has been a prime drug target of the pharmaceutical industry due to its sequence and structural distinction from human proteases; multiple selective inhibitors against the “N53” cis-protease are currently in use for treating HCV infections worldwide, and several new compounds are currently under evaluation by the FDA.

As demonstrated herein, Notch-signaling can be made drug-dependent by insertion of a protease domain, such as the NS3 protease domain of SEQ ID NO: 32 or variants thereof, between the extracellular and transmembrane domains of the Notch ligand Delta or “DLL” (FIGS. 6A-6C). In the absence of drug, the DLL extracellular domain is cleaved from its membrane anchor via NS3, releasing a soluble DLL ligand that is able to bind Notch but cannot activate it. Upon inhibition of NS3, however, DLL extracellular domain remains tethered to the cell surface and thus is able to activate Notch on the surface of opposing cells. This aspect of the technologies described herein has a variety of applications in biology and medicine, and is a powerful tool for developmental signaling, cancer biology, T cell engineering, and tissue engineering. In some embodiments, this system is used to achieve drug-control over “synthetic Notch” receptors in which the native Notch ligand-binding domain is substituted with an alternative protein (such as, e.g., anti-GFP scFv) for activation by cells expressing complementary surface ligands (such as, for example, GFP).

Synthetic proteins (or constructs expressing such synthetic proteins) comprising a receptor ligand (DLL, or any other ligand which binds to the target receptor), NS3 domain, and a targeting domain (e.g., an antibody specific for a cancer cell antigen) can be used, in some embodiments, for therapeutic applications. Once bound to their target (e.g., a cancer cell), these constructs would only activate receptors on adjacent cells when in the presence of an NS-inhibitor drug. Absent the drug, the NS3 domain would be cleaved, releasing the ligand and preventing receptor activation.

In some embodiments, the receptor ligand domain or extracellular domain is the extracellular domain of gamma secretase, nicastrin. Gamma secretase is a membrane protein complex involved in biological functions such as Notch and amyloid precursor protein (APP) processing. Its proteolytic subunit, presenilin, acts by catalyzing the cleavage of intramembrane alpha helices, and in turn allows the release of both the extracellular domain (important in APP pathology) and the intracellular domain (important in Notch developmental biology). The gamma secretase extracellular subunit, nicastrin, has been shown to regulate this process through steric hindrance. Gamma secretase substrates with bulky extracellular domains are resistant to proteolysis, and the regulated shedding of this bulky ectodomain is a key pathway in Notch processing.

The drug-controllable ligands can be used, in some embodiments, with various combinations of other aspects described herein, including the cis-clamp and autoinhibitory Notch receptors.

Also provided herein in some aspects, are modified HCV NS3 protease domains, as versatile protein engineering modules that can be applied to install drug-sensitivity into both intracellular and cell-surface proteins. In its natural context, NS3 is a serine cis-protease that excises itself from the HCV polyprotein by cleaving recognition sites that flank it at either end.² Because it is essential for HCV replication, numerous inhibitors targeting the viral protease have been developed. In previous work, NS3 and its inhibitors have been combined to create tools for conditionally linking proteins to imaging tags and degradation sequences.³⁻⁶ Given its successful application in these non-natural contexts, it was determined whether the viral protease could be used to design synthetic and drug-sensitive proteins to gain control over complex cellular processes such as transcription and intercellular signaling.

NS3 was tested as a module for engineering drug-sensitive transcription factors (TFs), using the protease as a Ligand-Inducible Connection (LInC) to control the association between modular DNA-binding (DB) and transcriptional activation (TA) domains. In some embodiments, NS3 was inserted in between minimal DB and TA sequences sourced from the yeast TF Gal4, generating Gal4_(DB)-NS3-Gal4_(TA) (FIG. 15A). In this configuration, it was expected that the viral protease would serve as a self-immolating linker, excising itself from the fusion construct and, in doing so, separating the DB and TA elements. However, in the presence of an NS3 inhibitor, it was believed that self-excision of the protease would be blocked, resulting in the preservation of full-length TF capable of activating the expression of targeted genes.

To determine whether DBGal4-NS3-TAGal4 behaved in this manner, the protein was stably expressed in a mammalian cell line containing a Gal4-dependent fluorescent reporter construct (UAS H2B-Citrine). Immunoblotting confirmed that intact DBGal4-NS3-TAGal4 accumulated in cells treated with the selective NS3 inhibitor BILN-2061, while full-length TF was not detected in drug-untreated controls (FIG. 15B). In addition to TF stabilization, analyses by fluorescence imaging, flow cytometry, and antibody detection showed that treatment with NS3 inhibitors also induced expression of H2B-Citrine in a dose-dependent manner (FIGS. 15B-15C). Various commercially available NS3 inhibitors were evaluated and multiple compounds capable of inducing robust transcriptional responses were identified, including BILN-2061, asunaprevir, danoprevir, and grazoprevir (FIG. 18). It was noted that the α-ketoamide-based inhibitors tested (telaprevir and boceprevir) did not induce detectable levels of reporter expression above background. Together, these results indicate that NS3 inhibitors can be used to precisely regulate the association of protease-linked modules in order to achieve inducible control over gene expression.

“NS3 inhibitors,” as used herein, refer to inhibitors of NS3 protease domain activity. Typically, NS3 protease inhibitors have been classified into two groups. (1) The first generations inhibitors (boceprevir and telaprevir) are linear a-ketoamide derivatives. These two inhibitors form a covalent bond with the active site of the enzyme in a reversible way. The second generation of inhibitors are mostly linear and macrocyclic noncovalent inhibitors of the NS3-4A enzyme. Accordingly, in some embodiments, non-limiting examples of NS3 inhibitors that can be used with the synthetic protein compositions and methods described herein comprising NS3 protease domains, such as SEQ ID NO: 32, or variants thereof, include paritaprevir, grazoprevir, CH 503034 (Boceprevir), VX-950 (Telaprevir), BI 201335, SCH 900518 (Narlaprevir), SCH6 (SCH446211), BILN 2061 (Ciluprevir), TMC435 (Simeprevir), ITMN-191/RG7227 (Danoprevir), MK-7009 (Vaniprevir),GS-9256, ACH-1625, MK-5172, ABT-450, IDX320, BMS-650032 (Asunaprevir), ACH-806 (GS-9132), and PHX1766.

Given the modular framework of the system, it was tested whether different transcription factors possessing tailored properties could be readily engineered. For example, it was tested whether substitution of the Gal4 TA domain with more potent transcriptional effectors (such as VP64, VP64-p65, and VPR)⁷ would yield TFs with enhanced drug sensitivity. Indeed, DBGal4-NS3-TAVP64-p65 and DBGal4-NS3-TAVPR resulted in higher reporter expression at decreased drug concentrations (including robust expression at drug concentrations in the low nanomolar range) as compared to the initial DBGal4-NS3-TAGal4 (FIG. 15D). Additionally, TFs with activity against alternative promoters were also designed, including one in which the reverse tetracycline repressor (rTetR)⁸ was used as the DB element (DBrTetR-NS3-TAVP64-p65). In transfected cells, DBrTetR-NS3-TAVP64-p65 exhibited “AND” gate activity, requiring both the presence of doxycycline (to induce rTetR binding to tetO sequences) and an NS3 inhibitor in order to activate transcription from the tetO-containing TRE promoter (FIG. 19). Notably, the effects of TF preservation and gene activation were reversed following inhibitor removal (FIG. 28).

To complement the “turn-on” systems described above, a strategy in which NS3 inhibitors could be used to “turn-off” gene expression was also designed for use in other aspects. In this approach, the NS3 protease was used to conditionally link an intact Gal4 (Gal4min) TF to a membrane-targeting domain with the expectation that protease inhibitor could be used to precisely regulate the amount of soluble versus membrane-bound TF. Using a Type-I transmembrane protein as a targeting element, a fusion construct was generated containing NS3 and Gal4min as a C-terminal cytosolic domain (TMD-NS3-Gal4min) (FIG. 15E).

Fluorescence imaging of cells expressing a dual-tagged version of the protein (BFP-TMD-NS3-Gal4_(m)i_(n)-mCherry) showed that Gal4_(min)was released from its BFP-fused transmembrane domain in drug-untreated cells, resulting in a liberated TF unit (tagged with mCherry) that localized predominantly to the nucleus (FIG. 15F). However, in cells in which NS3 activity had been inhibited, the TF remained linked to its targeting element and thus trafficked to endoplasmic reticulum (ER) surface and plasma membrane (PM). A version in which an N-terminal myristoylation and palmitoylation substrate⁹ was used as the targeting domain (myr-palm-NS3-Gal4min) exhibited similar behavior, becoming occluded from the nucleus in drug-treated cells (FIGS. 15G-15H).

Given that TFs must localize to the nucleus in order to bind their DNA targets, whether TMD-NS3-Gal4min and myr-palm-NS3-Gal4min would facilitate target gene expression in reporter cells that could be inducibly downregulated through NS3 inhibition was tested. Confirming these were immunoblotting and flow cytometry analyses showing that constructs could be used to achieve drug-inducible suppression of a Gal4-dependent fluorescent reporter gene (FIG. 20). Consistent with these results were analyses showing that exposure to NS3 inhibitors led to the accumulation of membrane-tethered Gal4min (FIGS. 15F-15G, 20A and 20B), as well as the gradual depletion of previously-cleaved TF copies (FIGS. 29A and 29B). Measurements by flow cytometry confirmed that drug treatment suppressed reporter gene expression in a dose-dependent manner (FIGS. 20A and 20B), and live-cell imaging showed that the effect of downregulation could be reversed following inhibitor withdrawal. Together, these results demonstrate that TFs can be conditionally linked to localization signals through NS3, in turn permitting precise control over their spatial distributions and activities.

Recognizing that natural gene regulation often involves the synchronized regulation of multiple genes, the “turn-on” and “turn-off” systems were combined to create a platform for simultaneously regulating distinct promoters using drug. In cells that coexpressed both a “turn-on” TF and “turn-off” TF (DBrTetR-NS3-TAVP64-P65 and TMD-NS3-Gal4_(m)i_(n), respectively), coinciding and inverse regulation of TRE- and UAS-controlled reporter genes were observed (FIG. 15I). These results demonstrate NS3 can be used to control multiple TFs in individual cells to activate concurrent and inverse gene expression changes in response to NS3 inhibition. Such strategies provide powerful approached for generating sophisticated, drug-dependent genetic circuits for programming complex behaviors into therapeutic mammalian cells.

In addition to TFs targeting engineered promoters, in some aspects, provided herein are drug-sensitive proteins or “synthetic drug-dependent proteins” that can be used to upregulate gene expression from endogenous promoter sequences, such as the synthetic drug-dependent protein of SEQ ID NO: 45 or engineered variants thereof. To achieve such control, as described herein, NS3 was integrated into artificial TFs based on dCas9, a catalytically inactive mutant of the Cas9 nuclease that can serve as a programmable DNA-binding domain.¹⁰⁻¹¹ First, a LInC module (e.g., NS3 protease domain) was integrated into dCas9-VPR¹³ in between the DB scaffold and a C-terminal region containing a nuclear localization sequence (NLS) and the VPR TA element (dCas9-NS3-NLS/VPR) (FIG. 16A). In this configuration, it was tested whether NS3 cleavage would not only inactivate the TF, but also prevent cleaved dCas9 from translocating into the nucleus (e.g., would be cytoplasmically contained).

Western blotting of cell lysates demonstrated that full-length copies of dCas9-NS3-NLS/VPR accumulated only in cells cultured in the presence of drug (FIG. 16B), and fluorescence imaging of immunostained cells indicated that the dCas9 domain localized to the nucleus in a drug-dependent manner (FIG. 16C, FIG. 21). In addition, live-cell time-dependent dye labeling experiments carried out using a SNAP-tag fused version of the TF (SNAP-dCas9-NS3-NLS/VPR) showed that only protein copies made in N53-inhibited cells were transported across the nuclear envelope (FIG. 16D). Given that unfused dCas9 molecules can serve as inhibitors of native expression levels (by binding and occupying targeted DNA sites)¹², the cytoplasmic containment of cleaved dCas9 could serve to prevent undesired gene repression in drug-untreated cells, as the unfused domain has been reported to suppress gene expression in certain cases through binding and occupying targeted DNA sites.

To confirm that dCas9-NS3-NLS/VPR can be used to upregulate gene expression in a drug-inducible manner, the TF was co-expressed with sgRNA sequences targeting either a fluorescent reporter construct (UAS H2B-Citrine), or a chromosomal region upstream of the human gene encoding the chemokine receptor CXCR4.¹³ Flow cytometry analyses showed that BILN-2061 can be used to induce upregulation of both gene targets in a dose-dependent manner (FIG. 16E, FIG. 22). In addition, tests using separate sgRNAs targeting distinct regions of the CXCR4 promoter showed that (under saturating drug concentrations) dCas9-NS3-NLS/VPR was able to upregulate receptor expression to a similar extent as dCas9-VPR (FIG. 23). A system in which NS3 was used to regulate TA domain association with a hairpin-modified sgRNA¹³ was also developed (FIG. 24). Together, these data indicate that NS3 can also be combined with dCas9 to achieve tunable transcription of endogenous human genes, and also suggest that other sophisticated Cas9-based tools¹⁴ could be designed using a similar approach.

In addition to TFs, certain transmembrane signaling proteins are also known to possess component-based architecture, including the Notch receptor, its ligands, and their synthetic derivatives.¹⁵⁻¹⁷ Thus, it was tested whether NS3 can also be used to regulate intercellular signaling via drug-dependent Notch/SynNotch activation. Toward this end, an NS3-containing version of the Notch ligand Delta-like 1 (Dll1) was designed by integrating the protease into the extracellular portion of the protein (Dll1-NS3), positioning it between the receptor-binding region and transmembrane domain (TMD) (FIG. 17A). In this configuration, it was tested whether NS3 self-excision would yield a soluble ligand that, due to its lack of a membrane tether, would not be presented at cell-surface (FIG. 17B). Indeed, immunostaining of cells stably expressing Dll1-NS3 showed that presentation of Dll1-NS3 at the cell surface was induced upon NS3 inhibition (FIG. 17C).

In the prevailing model of Notch activation, the endocytosis of membrane-tethered ligand is thought to deliver a mechanical “pulling” energy that is required to trigger the “on” state of the receptor, in turn inducing the release of its intracellular domain (NICD, a transcriptional effector). Because Notch activation requires the endocytosis of membrane-tethered ligands,¹⁸ it was tested whether drug-preserved Dll1-NS3 copies would be able to mediate trans-cellular signaling. To determine whether protease-containing ligand could activate Notch signaling in a drug-dependent manner, “sender” cells expressing Dll1-NS3 were combined with Notch1-expressing “receiver” cells in a coculture assay (FIG. 17D). Using receiver cells containing a NICD-dependent fluorescent reporter gene (12×CSL H2B-Citrine)¹⁹, Notch activation at sender cell-receiver cell interfaces was observed only in drug-treated cocultures (FIGS. 17E-17F, FIG. 25). Thus, in addition to serving as a versatile module for controlling intracellular proteins, these results demonstrate that NS3 can also be applied in luminal and cell-surface contexts to regulate cell-cell recognition events and in turn control intercellular communication.

The applications described herein demonstrate that the HCV NS3 protease of SEQ ID NO: 32 is a versatile domain that can be used to straightforwardly engineer drug-sensitivity into both intracellular and cell-surface proteins. Through the implementation of simple and intuitive protein designs, tightly-regulated chemical control over complex cellular phenomena was achieved. One significant advantage of the methods described herein is the availability of highly-selective NS3 inhibitors, many of which have been tested for clinical use and can be obtained from commercial sources. Thus, in addition to its potential applications in basic biology investigations, the methods described herein can also serve as a powerful strategy for regulating therapeutic cells in vivo using safe and clinically-approved antiviral drugs.

In some embodiments, the HCV NS3 protease domain and corresponding recognition sites can be substituted with other protease domains and recognition sites from other viruses including, but not limited to, human immunodeficiency virus and human rhinovirus.

In those embodiments of the synthetic or recombinant proteins described herein where one or more of the protein domains is mutated or engineered or modified relative to the endogenous or naturally occurring protein, such as a mutated Notch Negative Regulatory Region (NRR), for such purposes as enhancing binding or efficacy, or stability, techniques known in the art for identifying mutated proteins or domains having one or more desired properties can be used. For example, modified or mutated domains or polypeptides can be produced, for instance, by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) does not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.

Naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Preferred conservative substitutions for use in the synthetic proteins described herein are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu. Whether a change in the amino acid sequence of a synthetic protein results in a functional variant can be readily determined by assessing the desired activity of the variant synthetic protein or polypeptide relative to the non-mutated version of the synthetic protein.

As known to those of skill in the art, receptors tend to have three regions or domains: an extracellular domain for binding ligands such as proteins, peptides or small molecules, a transmembrane domain that traverses the cellular membrane, and an intracellular or cytoplasmic domain which frequently can participate in some sort of signal transduction event within a cells, such as phosphorylation. Accordingly, in some embodiments of the aspects described herein, the synthetic proteins can comprise various combinations of extracellular, transmembrane, and intracellular domains derived from naturally occurring domains or engineered versions of such domains. Non-limiting examples of transmembrane domains that can be used with the synthetic proteins described herein include SEQ ID NOs: 13, 14, and 31, and engineered or mutant variants thereof. Non-limiting examples of intracellular domains that can be used with the synthetic proteins described herein include the Notch Intracellular Domain (NICD) of SEQ ID NOs: 11, and engineered or mutant variants thereof.

It is also understood that different elements or domains of the synthetic proteins can be arranged in any manner that is consistent with the desired functionality. For example, a synthetic, drug-dependent protein can comprise an extracellular or ligand binding domain (LBD), such as SEQ ID NO: 2 or an engineered variant thereof, an NS3 protease domain of SEQ ID NO: 32 or variant thereof, and a transmembrane domain from N-terminal to C-terminal, in some embodiments. In some embodiments, additional domains or amino acid sequences can be included C- or N-terminal to the various domains comprising the synthetic proteins described herein.

In some embodiments of the aspects described herein, a synthetic protein comprises one or more domains from or derived from a transcriptional regulator. Transcriptional regulators either activate or repress transcription from cognate promoters. Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Transcriptional repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase. Other transcriptional regulators serve as either an activator or a repressor depending on where it binds and cellular conditions. Accordingly, as used herein, a “transcriptional activation domain” refers to the domain of a transcription factor that interacts with transcriptional control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to increase and/or activate transcription of one or more genes. Non-limiting examples of transcriptional activation domains include: a herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP16), HIV TAT, a NF B p65 activation domain, p53 activation domains 1 and 2, a CREB (cAMP response element binding protein) activation domain, an E2A activation domain, NFAT (nuclear factor of activated T-cells) activation domain, yeast GAL4, yeast GCN4, yeast HAP1, MLL, RTG3, GLN3, OAF1, PIP2, PDR1, PDR3, PHO4, LEU3 glucocorticoid receptor transcription activation domain, B-cell POU homeodomain protein Oct2, plant Ap2, or any others known to one or ordinary skill in the art. A transcriptional activation domain can comprise a wild-type or naturally occurring sequence, or it can be a modified, mutant, or derivative version of the original transcriptional activation domain that has the desired ability to increase and/or activate transcription of one or more genes. In some embodiments, transcription activation domains for use with the synthetic proteins described herein are selected from Gal4, VP64, VP64-p65, and VPR reverse tetracycline repressor.

In some embodiments of the aspects described herein, a synthetic protein comprises one or more “DNA-binding domains” (or “DB domains”). Such “DNA-binding domains” refer to sequence-specific DNA binding domains that bind a particular DNA sequence element. Accordingly, as used herein, a “sequence-specific DNA-binding domain” refers to a protein domain portion that has the ability to selectively bind DNA having a specific, predetermined sequence. A sequence-specific DNA binding domain can comprise a wild-type or naturally occurring sequence, or it can be a modified, mutant, or derivative version of the original domain that has the desired ability to bind to a desired sequence. In some embodiments, the sequence-specific DNA binding domain is engineered to bind a desired sequence. Non-limiting examples of proteins having sequence-specific DNA binding domains that can be used in synthetic proteins described herein include GAL4, GCN4, reverse tetracycline receptor, THY1, SYN1, NSE/RU5′, AGRP, CALB2, CAMK2A, CCK, CHAT, DLX6A, EMX1, zinc finger proteins or domains thereof, CRISPR/Cas proteins, such as Cas9, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu196 , and TALES.

In those embodiments where a CRISPR/Cas-like protein is used, the CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein. The CRISPR/Cas-like protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of the CRISPR/Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR/Cas-like protein can be truncated to remove domains that are not essential for the functions of the systems described herein. In some embodiments of the engineered systems, methods, and compositions thereof disclosed herein, a CRISPR enzyme that is used as a DNA binding protein or domain thereof is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR or domain thereof lacks the ability to cleave a nucleic acid sequence containing a DNA binding domain target site. For example, in some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.

In some embodiments of the synthetic proteins described herein, one or more additional “fusion” domains can be added to the synthetic protein to provide additional desired functionality. Well known examples of fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain can be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HIS6 (SEQ ID NO: 69)) fusion partners. As another example, a fusion domain can be selected so as to facilitate detection of the synthetic proteins. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. Non-limiting tag sequences that can be used in the synthetic proteins described herein include SEQ ID NOs: 5-7 and 33. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation. Other types of fusion domains that can be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domain.

In some embodiments, a cell is transfected or transformed with a nucleic acid sequence encoding the synthetic protein of interest.

As used herein, the term “transfection” is used to refer to the uptake of an exogenous nucleic acid by a cell, and a cell has been “transfected” when the exogenous nucleic acid has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids into suitable host cells.

Suitable techniques of transfection for use with the compositions and methods described herein include, but are not limited to calcium phosphate-mediated transfection, DEAE-dextran mediated transfection, and electroporation. Cationic lipid transfection using commercially available reagents including the Boehringer Mannheim Transfection Reagent (N.fwdarw.1-(2,3-Dioleoyloxy)propyl-N,N,N-trimethyl ammoniummethylsulfate, Boehringer Mannheim, Indianapolis, Ind.) or LIPOFECTIN or LIPOFECTAMIN or DMRIE reagent (GIBCO-BRL, Gaithersburg, Md.) can also be used.

The term “transformation” as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transfection, the transforming nucleic acid can recombine with that of the cell by physically integrating into a chromosome of the cell, can be maintained transiently as an episomal clement without being replicated, or can replicate independently as a plasmid. A cell is considered to have been stably transformed when the transforming nucleic acid is replicated with the division of the cell.

As used herein an “expression vector” refers to a DNA molecule, or a clone of such a molecule, which has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that would not otherwise exist in nature. DNA constructs can be engineered to other domains operably linked to nucleic acid segments encoding a desired synthetic or recombinant protein of interest. In addition, an expression vector can comprise additional DNA segments, such as promoters, transcription terminators, enhancers, and other elements. One or more selectable markers can also be included. DNA constructs useful for expressing cloned DNA segments in a variety of prokaryotic and eukaryotic host cells can be prepared from readily available components or purchased from commercial suppliers.

Expression vectors can also comprise DNA segments necessary to direct the secretion of a polypeptide or protein of interest. Such DNA segments can include at least one secretory signal sequence. Secretory signal sequences, also called leader sequences, prepro sequences and/or pre sequences, are amino acid sequences that act to direct the secretion of mature polypeptides or proteins from a cell. Such sequences are characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins. Very often the secretory peptide is cleaved from the mature protein during secretion. Such secretory peptides contain processing sites that allow cleavage of the secretory peptide from the mature protein as it passes through the secretory pathway. A recombinant protein of interest can contain a secretory signal sequence in its original amino acid sequence, or can be engineered to become a secreted protein by inserting an engineered secretory signal sequence into its original amino acid sequence. The choice of suitable promoters, terminators and secretory signals is well within the level of ordinary skill in the art. Expression of cloned genes in cultured mammalian cells and in E. coli, for example, is discussed in detail in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor, N.Y., 2012; which is incorporated herein by reference in its entirety). Non-limiting examples of signal sequences for use with the synthetic proteins described herein include SEQ ID NOs: 1 and 41.

After transfection, the host cell can be maintained either transiently transformed or stably transformed with said nucleic acid or expression vector. Introduction of multiple nucleic acids or expression vectors, and selection of cells containing the multiple nucleic acids or expression vectors can be done either simultaneously or, more preferably, sequentially. The technique of establishing a cell line stably transformed with a genetic material or expression vector is well known in the art (Current Protocols in Molecular Biology). In general, after transfection, the growth medium will select for cells containing the nucleic acid construct by, for example, drug selection or deficiency in an essential nutrient, which is complemented by a selectable marker on the nucleic acid construct or co-transfected with the nucleic acid construct. Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free medium. Selection of a medium appropriate for the particular host cell used is within the level of ordinary skill in the art.

Suitable selectable markers for drug selection used with the compositions and methods described herein include, but are not limited to, neomycin (G418), hygromycin, puromycin, zeocin, colchine, methotrexate, and methionine sulfoximine.

A cell to be engineered with synthetic proteins or combinations thereof described herein can be any cell or host cell. As defined herein, a “cell” or “cellular system” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular. A “natural cell,” as defined herein, refers to any prokaryotic or eukaryotic cell found naturally. A “prokaryotic cell” can comprise a cell envelope and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions.

In some embodiments, the cell is a eukaryotic cell. A eukaryotic cell comprises membrane-bound compartments in which specific metabolic activities take place, such as a nucleus. In other embodiments, the cell or cellular system is an artificial or synthetic cell. As defined herein, an “artificial cell” or a “synthetic cell” is a minimal cell formed from artificial parts that can do many things a natural cell can do, such as transcribe and translate proteins and generate ATP.

Once a drug resistant cell population is established, individual clones may be selected and screened for high expressing clones. Methods of establishing cloned cell line are well known in the art, including, but not limited to, using a cloning cylinder, or by limiting dilution. Expression of the recombinant protein of interest from each clone can be measured by methods such as, but not limited to, immunoassay, enzymatic assay, or chromogenic assay. A cell line stably transformed with a first nucleic acid construct may be then used as host cell for transfection with a second or more nucleic acid constructs, and subjected to different drug selections.

By “cell culture” or “culture” is meant the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, can be used.

As used herein, “cell culture medium” is a media suitable for growth of animal cells, such as mammalian cells, in in vitro cell culture. Cell culture media formulations are well known in the art. Typically, cell culture media are comprised of buffers, salts, carbohydrates, amino acids, vitamins and trace essential elements. “Serum-free” applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum. Various tissue culture media, including defined culture media, are commercially available, for example, any one or a combination of the following cell culture media can be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL.TM. 300 Series (JRH Biosciences, Lenexa, Kans.), among others. Serum-free versions of such culture media are also available. Cell culture media can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured and/or the desired cell culture parameters.

As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation. Accordingly, the terms “comprising” means “including principally, but not necessary solely”. Furthermore, variation of the word “comprising”, such as “comprise” and “comprises”, have correspondingly the same meanings. The term “consisting essentially of” means “including principally, but not necessary solely at least one”, and as such, is intended to mean a “selection of one or more, and in any combination”. Stated another way, the term “consisting essentially of” means that an element can be added, subtracted or substituted without materially affecting the novel characteristics of the invention. This applies equally to steps within a described method as well as compositions and components therein. In other embodiments, the inventions, compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (“consisting of”). For example, a composition that comprises elements A and B also encompasses a composition consisting of A, B and C.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, publications, and websites identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

EXAMPLES Example 1

The Notch protein is a transmembrane receptor that acts a mechanical “switch,” translating mechanical cues into gene expression. This mechanosensing activity is achieved via Notch's force-sensitive Negative Regulatory Region, which contains three LNR domains. In the resting state, the LNR domains adopt an autoinhibitory conformation that sterically hinders proteolytic cleavage necessary for receptor activation. Upon the application of a pulling force, however, these LNR domains are displaced, and two concomitant proteolytic cleavages occur that release the Notch intracellular domain to transport to the nucleus and regulate gene expression. Synthetic notch receptors have been created that allow for gene expression to be controlled upon binding of the receptor to various ligands of interest (e.g. surface proteins on cancer cells).

Described herein are strategies through which signaling form natural and synthetic Notch receptors, as disclosed in US patent Application 2016/0264665, which is incorporated herein in its entirety by reference, can be regulated/modulated using antibody domains. These systems are used to increase the amount of force required to activate Notch receptors, or to regulate their activity on therapeutic cells. The described tools are useful for a variety of cell engineering applications, including the creation of engineered cells capable of sensing certain mechanical features of solid tumors (or biomaterials), as well as permit the precise control over therapeutic/engineered cells expressing synthetic Notch receptor proteins.

These systems involve the use of antibody fragments directed against the NRR region of the Notch receptor, a force-sensitive mechanical switch that is ruptured during receptor activation. Binding of these antibodies stabilizes the NRR and prevents Notch activation. Use of scFvs from these antibodies will permit the generation of inhibitory “modules” which we will use to generate synthetic proteins and receptors to precisely control signaling from Notch/SynNotch systems.

Antibody fragments from anti-NRR antibodies are used as new modules for engineering synthetic proteins and receptors for cell-engineering applications. All previous work involving anti-NRR antibodies have relied on the use of purified immunoglobulin as an exogenously applied drug/agent. In the work described herein, these antibody-derived fragments are used as new “genetic tools” to reprogram natural and synthetic Notch signaling for synthetic biology applications. This allows cells expressing these systems to function as genetically encoded “tensometers,” permitting, for example, engineered T cells to activate their cell killing activity in response to the mechanical properties of fibrotic tissues or physical features of solid tumors.

Work described herein is also directed towards constructs and methods for controlling the binding/activation of notch receptors through the use of synthetic ligands that incorporate NS3 protease domains from the hepatitis C virus. In the absence of an NS-inhibitor drug, the ligand domain is cleaved and becomes incapable of activating the notch receptor. When an NS-inhibitor drug is applied, the NS3 domain remains intact and the ligand is capable of activating the notch receptor.

Non-limiting applications for the compositions, systems, and methods thereof described herein include:

-   -   a) SynNotch receptors with defined/programmable force-activation         thresholds for applications in cell engineering, T cell         immunotherapy, and tissue engineering.     -   b) Regulating SynNotch proteins and reducing their background         levels of activity. In principle, genetic regulation of         cis-clamps could be used to “turn off” the cell-killing activity         of immune cells.     -   c) Drug-inducible control of notch/synNotch activation

Advantages of the compositions, systems, and methods thereof described herein include:

-   -   a) Receptors with increased force activation thresholds are         completely novel; potential applications are wide-ranging,         including generation of cells with ability to detect physical         features of solid tumors, mechanical properties of biomaterials,         etc.     -   b) cis-clamps will permit regulation on engineered cells         expressing SynNotch receptors (including therapeutic T cells)         and can be useful for reducing the known background activity of         these receptors.     -   c) Drug-inducible notch activation that allows tighter control         of therapeutic interventions that utilize notch receptor         transduction mechanisms

Notch Cis-Clamps

Some embodiments of the aspects described herein pertain to “cis-clamps” which are synthetic proteins that comprise an NRR-binding scFv fused to a transmembrane domain. Notch is activated by ligands expressed on adjacent cells, but inhibited when ligands are expressed on the same cell through a mechanism known as “cis-inhibition”, as shown in FIG. 1A. This cis-interaction serves to prevent cells from receiving signals from their neighbors, and also prevents spontaneous “ligand independent” background activation, reducing Notch background activity.

Cis-inhibitors are useful for cell engineering, including, for example, therapeutic T cells, as Notch/SynNotch receptors are known to exhibit background “leaky” activation. Membrane-tethered anti-NRR scFvs as genetically encoded Notch inhibitors, or “cis-clamps” as utilized here (FIG. 1B). These scFvs are derived from antibodies that are known to bind and stabilize the NRR region of Notch receptors, preventing their activation. These “clamps” can be used to regulate Notch/SynNotch activity in a manner similar to ligand cis-inhibition. In engineered cells, these chimeras can be used as regulatory elements to limit signaling from synthetic Notch receptors and, for instance, reduce off-target T cell killing in the case of engineered SynNotch T cells. As demonstrated in FIG. 12, these genetically encoded inhibitors can be placed under the control of a drug-inducible system, allowing temporal control over SynNotch T cell activity.

cis-clamps are especially useful in situations where ligand co-expression is problematic. The most obvious route toward cis-inhibition of a SynNotch receptor is to co-express the receptor and its target ligand on the same cell. However, in the case of cell immunotherapy, ligands used to activate SynNotch receptors on T cells are usually cell surface cancer markers. While co-expression of these markers would in principle permit cis-inhibition of SynNotch proteins, they could also cause the therapeutic cells to attack one another. Thus, use of cis-clamps provides a novel route through which SynNotch receptors can be regulated without the introduction of cancer marker/antigens to the engineered cells.

Without wishing to be bound or limited by theory, the work described herein indicates that tuning the affinity of the scFv allows for the engineering of mechanical sensitivity. Data presented in FIGS. 2A-2C support this and shows that NRR-binding scFv is mutated and expressed as a separate transmembrane cis-clamp. Strategies for tuning scFv affinity involve, for example, studying available crystal structures of the Notch1 NRR bound to the scFv (Wu et al, 2010) to make structure-guided residue mutations. Amino acid residues on the scFv complementarity-determining regions (CDRs) that tightly interact with the NRR are identifiable targets for engineering, where predictions can be made to substitute residues that will slightly to substantially modify scFv affinity, as shown in FIGS. 2A-2C. Alternatively, instead of targeted mutations, random mutagenesis of these CDRs through techniques such as, for example, error-prone PCR, can also be used to perform the directed evolution of mechanical sensitivity. Other sites within the scFv provide opportunity for tuning include disulfide bridges, alteration of which has been shown to affect overall scFv stability.

Auto-Inhibitory Notch Receptor

Another aspect of the compositions, systems and methods described herein is directed towards synthetic auto-inhibitory notch receptors that contain an NRR-binding scFv. As in the cis-clamp embodiment, the scFv in the auto-inhibitory notch receptor stabilizes the NRR region resulting in more force being required to activate the receptor.

NRR-binding scFv's is expressed as a contiguous part of Notch-based receptors to act as a synthetic “fourth LNR domain” (LNR4), offering additional stability to the NRR and increasing the force threshold of Notch activation. An example of this approach is described in FIGS. 3A and 3B.

The LNR4 domain can also be mutated to change the affinity of binding to the NRR in order to tune the mechanical sensitivity of the receptor, as previously demonstrated in FIG. 2A for the cis-clamp embodiment.

Notch Constructs With Synthetic NRR Domains

Another aspect of the compositions, systems and methods described herein is directed towards synthetic notch receptors with mutated NRR domains. Mutating the NRR domain and utilizing an scFv that has high affinity to the mutated NRR (but not the native NRR) in either the cis-clamp or auto-inhibitory receptor configurations allows for a more specific system with reduced off-target effects (e.g., the scFv binding the NRR region on notch receptors of adjacent cells).

One strategy for creating these synthetic NRR-scFv pairs is to design a chimeric NRR domain that contains components from both the Notch1 and Notch2 NRR domains. Similar to scFv that binds NRR1, scFv's that bind and inhibit NRR2 are generated. It has been demonstrated that these scFv's are specific to their respective NRR domains and that they are unable to bind chimeric NRR1-NRR2 domains with intermixed components. Seeing as these scFv's are specific to each NRR and insensitive to certain NRR chimeras, in some embodiments, design of a reverse chimera, for example, an αNRR1-αNRR2 scFv fusion that is specific to an NRR chimera but insensitive to WT NRR1 and NRR2. Such an scFv can be designed through structure-guided decisions based off the NRR-scFv crystal structure and amino acid alignments between the two NRRs and scFvs, or it can be generated through traditional methods of antibody evolution and purification. A synthetic NRR-scFv pair such as this allows scFv-based regulation of SynNotch activity that is orthogonal to native Notch signaling. FIGS. 13A-13C provides more details on this approach.

Drug-Controllable Notch Signaling

Yet another aspect of the compositions, systems and methods described herein is directed towards constructs and methods for controlling the binding/activation of notch receptors through the use of synthetic ligands that incorporate NS3 protease domains from the hepatitis C virus (HCV). The NS3 domain is a serine protease embedded within the HCV polyprotein that excises itself from the precursor polypeptide by cleaving recognition sites flanking it at either end. The enzyme has been a prime drug target of the pharmaceutical industry due to its sequence and structural distinction from human proteases. Multiple selective inhibitors against the “NS3” cis-protease are currently in use for treating HCV infections worldwide, and several new compounds are currently under evaluation by the FDA.

As described herein, in some embodiments, Notch-signaling can be made drug-dependent by insertion of the NS3 protease domain between the extracellular and transmembrane domains of the Notch ligand Delta (DLL, FIGS. 6A-6C). In the absence of drug, the DLL extracellular domain is cleaved from its membrane anchor via NS3, releasing a soluble DLL ligand that is able to bind Notch but cannot activate it. Upon inhibition of NS3, however, DLL extracellular domain remains tethered to the cell surface and thus is able to activate Notch on the surface of opposing cells. This technology has a variety of applications in biology and medicine, including, but not limited tom developmental signaling, cancer biology, T cell engineering, and tissue engineering. This system is applied, in some embodiments, to achieve drug-control over “synthetic Notch” receptors in which the native Notch ligand-binding domain is substituted with an alternative protein (such as anti-GFP scFv) for activation by cells expressing complementary surface ligands (such as GFP).

In addition, SynNotch activation can alternatively be made drug-dependent by incorporating the NS3 domain into the receptor rather than the ligand. For example, in some embodiments, an NS3 domain could be placed between the DNA binding domain (BD) and the activating domain (AD) of a Notch ICD transcription factor (such as Gal4). In the absence of drug, the ICD would lose its DNA-binding ability and become nonfunctional, whether or not a ligand on a target cell is bound. In the presence of drug, the ICD would remain intact and would regulate gene activity only upon ligand-induced receptor activation. FIG. 14 demonstrates feasibility of placing NS3 between the BD and AD of Gal4 to create a drug-dependent transcription factor with titratable activation. An NS3 domain can also be incorporated into the ECD of Notch, for example between the NRR and LBD, likewise creating a functional receptor only in the presence of an NS3-inhibitor.

Compositions, systems and methods described herein comprising a receptor ligand (DLL, or any other ligand which binds to the target receptor), NS3 domain, and a targeting domain (e.g. an antibody specific for a cancer cell antigen) can be used for a variety of therapeutic applications. Once bound to their target (e.g., a cancer cell), these constructs serve to activate receptors on adjacent cells only when in the presence of an NS-inhibitor drug. Absent the drug, the NS3 domain would be cleaved, releasing the ligand, LBD, or ICD BD and preventing gene regulation through receptor activation. The drug-controllable ligands and receptors can be used with various combinations of the previously described aspects, including the cis-clamp and autoinhibitory Notch receptors.

Example 2 Chemical and Physical Control Over Gene Expression in Mammalian Cells

Provided herein are compositions, systems and methods for measuring and managing biomolecules in living cells. These novel compositions, systems and methods described herein were developed for regulating gene expression in mammalian systems using chemical and physical cues. In addition to serving as valuable tools for studying numerous aspects of biology and disease, these technologies are powerful additions to the “toolkit” for engineering therapeutic cells.

Engineering cellular responses to mechanical cues. Cells sense forces via surface receptors that can directly affect changes in gene expression. Described herein is the use of transmembrane signaling protein Notch as a scaffold to engineer new, synthetic mechanoreceptors that are able to activate gene expression in response to defined and programmable amounts of applied force. These engineered receptors are used to design therapeutic mammalian cells capable of detecting various mechanical signals, such as matrix elasticity, or the unique physical features of solid tumors.

Remote control of therapeutic cells using FDA-approved antiviral drugs. Tools for regulating the therapeutic properties of engineered cells while they are in the body, and strategies to eliminate them from patients following a successful course of treatment, are necessary to ensure that cell-based therapeutics can be administered safely. The novel compositions, systems and methods described herein were created to control gene expression and signaling in therapeutic cells using FDA-approved antiviral small molecules, permitting in vivo control over these agents using safe, orthogonal, and orally-available drugs.

Engineering cellular responses to mechanical cues. Notch proteins regulate a variety of cell fate decisions and are activated through a mechanism involving mechanical force. In mammals, Notch receptors are single-pass transmembrane proteins containing a membrane-proximal domain known as the negative regulatory region (NRR, as shown in FIG. 7A). Biophysical and cellular studies have identified the NRR as a force-activated mechanical switch that serves to regulate the localization of the Notch intracellular domain (NICD), a transcriptional effector that is cleaved from the receptor and transported to the nucleus following activation (FIG. 7B).

In its quiescent state, the NRR adopts an autoinhibited conformation in which three LNR (LIN12-Notch repeat) modules sterically block a protease site (termed S2), which must be cleaved for receptor activation. Notch recognizes ligands of the Delta/Serrate/lag2 (DSL) and is activated upon the binding and endocytosis of ligands expressed by neighboring cells. The force applied to the receptor during ligand endocytosis serves to deliver a mechanical “pulling” energy that is able to displace the LNR modules and reveal the S2 site for cleavage by activating metalloproteases. Cleavage at S2 induces additional proteolysis at an intramembrane site (termed S3), which in turn causes the release of NICD from the plasma membrane, as shown in FIG. 7A.

Increasing the force threshold for Notch activation: Recent studies have suggested that molecular interactions within the NRR—specifically, the interactions made by and between the LNR modules—define the amount of force needed to activate Notch receptors. Without wishing to be bound or limited by theory, it is hypothesized that synthetic Notch proteins with increased activation thresholds can be created through stabilization of the autoinhibited conformation of the NRR.

In the work presented herein, human Notch-1 (hN1), a structurally well-characterized receptor that is activated by ≥5 picoNewtons of pulling energy, is used as a scaffold for engineering new mechanoreceptors. To increase hN1's force requirement, single chain variable fragments (scFvs) derived from anti-hN1 antibodies are introduced that are known to inhibit receptor activation by binding and stabilizing the NRR. These scFvs are integrated into the hN1 ectodomain at positions such that they are able to bind and stabilize their antigen (FIGS. 8A-8B), and previously reported x-ray structures of scFv:NRR complexes (FIG. 8C) are used to guide the selection of these positions.

These scFv-containing chimeras exhibit increased force requirements compared to their natural counterparts, as activation of these receptors requires the rupture of the scFv:NRR interaction in addition to that of the LNRs. Work described herein shows that an scFv-fused hN1 (hN1-scFv) is correctly processed in the Golgi apparatus and trafficked to the cell surface to a similar extent as the wild-type hN1 (FIG. 8D). Furthermore, several lines of evidence indicate that the integrated scFv is bound to the NRR within the receptor, and results also indicate that this binding interaction increases the force resistivity of the receptor.

Genetically encoded “tensometers” for sensing the mechanics of materials and solid tumors: Previous work has indicated that the forces required to rupture scFv:antigen complexes are correlated with their thermal dissociation rates, and that these forces can be predictably modulated via mutation.⁶ Thus, it is anticipated that scFv-containing Notch proteins should be highly susceptible to engineering, and that the activation thresholds of these receptors can be precisely tuned via design, or directed evolution. Single molecule force spectroscopy measurements using various immunoglobulin domains indicate that typical scFv:antigen rupture in response to forces ranging from 20 to 200 pN⁷—thus, it is possible to create Notch receptors with activation thresholds within this regime.

Reinforced Notch receptors have numerous applications in the study of cellular mechanosensation, as well as in the engineering of therapeutic mammalian cells. These receptors can be used to program cells to detect and execute specified gene expression programs in response to certain physical features within in their microenvironments. Indeed, work described herein shows that scFv-bound hN1 receptors are able to discriminate between ligands bound to stiff versus soft substrates, becoming activated only by ligands capable of applying sufficient tension to unbind the scFv from its fused antigen (FIGS. 9A and 9B). These findings show that scFv-containing Notch receptors can act as genetically encoded “tensometers,” permitting for example, engineered T cells to activate their cell killing activity in response to the mechanical properties of fibrotic tissues or physical features of solid tumors.

Furthermore, cells expressing a NRR-binding scFv exhibit distinct responses to magnetic fields (compared to hN1-expressing cells) when cultured in the presence of ligand-coated magnetic beads, raising the intriguing possibility that mammalian cells can be engineered to respond to different levels of magnetic force. To define these forces, single molecule measurements (using optical and magnetic tweezers) and biophysical assays (using DNA tension gauge tethers and reporter cells) are used to characterize the mechanics of individual receptors, and molecular dynamics are used to map the energy landscape of these receptors in response to varying degrees of applied energy.

Studies on Notch receptor trafficking and localization: Recombinant and endogenous hN1 is localized predominantly to the ER in a variety of mammalian cell lines. Expression of a membrane-tethered NRR-binding scFv (scFv-TMD) dramatically increases the surface localization of recombinant hN1 in CHO cells (FIG. 10), as well as the localization of endogenous hN1 in MCF-7 cells.

Furthermore, work described herein shows that low-affinity scFvs are able to increase the surface localization of hN1 without affecting its ability to respond to ligand-expressing cells. “Synthetic Notch” (syn-Notch) receptors (in which the native hN1 ligand-binding region is replaced with domains recognizing alternative targets, such as cancer markers), are used to engineer T cells for immunotherapy applications, thus, the described observations may have important implications for therapeutic cell engineering, as control over receptor localization and concentration are powerful strategies to regulate the activity of such systems.

Remote control of therapeutic cells using FDA-approved antiviral drugs. A major challenge associated with the development of cell-based therapies is the risk of unintended toxicities stemming from the residence of engineered cells within patients beyond the course of treatment. Thus, strategies for eliminating such cells—by inducing the expression of a “self-destruct” gene after successful treatment, for example—are necessary to ensure that cell-based therapeutics can be administered safely.

In recent work, researchers have begun to apply existing drug-inducible gene expression tools as regulatory systems for controlling therapeutic cells—however, many of these systems possess features that complicate their translation to clinical applications. For example, rapamycin and tamoxifen are widely used to control the activity of proteins fused to domains recognizing these drugs, but they are also ligands that bind and stimulate endogenous signaling proteins involved in regulating metabolism and immunity. Platforms based on the bacterially derived tetracycline-binding repressor proteins are similarly challenged by the mitotoxic properties of tetracyclines as well as their potent antibiotic activity. While the recently described inducible systems based on abscisic acid- and gibberellin-binding proteins have proven to be highly versatile, use in patients could be complicated by the abundance of these plant-derived hormones in the environment and in plant-based foods.

Inducible gene-expression using antiviral drugs: To circumvent the challenges imposed by the intrinsic bioactivity of these compounds, viral proteases and their corresponding anti-viral inhibitors are used herein to create new inducible systems that are orthogonal to mammalian systems. For example, the activity of diverse proteins can be made drug-sensitive by fusion with the hepatitis C virus (HCV) NS3 protease domain. NS3 is a serine cis-protease that excises itself from the HCV polyprotein by cleaving recognition sites flanking it at either end. Because it is essential for HCV replication, the NS3 protease has been a major target in the drug industry's development of anti-HCV therapeutics—thus, numerous inhibitors targeting this viral domain have previously been identified, including several that are FDA-approved.

NS3 protease domain can be used to render transcription factors (TFs) based on the nuclease-deficient dCas9 subject to drug-control. In an initial design strategy, NS3 is intergrated into the engineered TF dCas9-VPR, (generating dCas9-NS3-VPR, or “dCNV”) placing enzyme between the dCas9 scaffold and the “VPR” transactivation domain (TAD). In this configuration, the protease domain serves as a self-immolating linker that leads to the dismemberment of the artificial TF, separating dCas9 from its fused nuclear localization signal (NLS) and VPR motif. However, upon exposure to an NS3 inhibitor, self-excision of the protease is blocked and intact copies of the TF are in turn able to transport into the nucleus to activate the expression of targeted genes. Using an sgRNA sequence complementary to the promoter region of chromosomal DNA encoding the chemokine receptor CXCR4, dCNV was able to activate upregulate expression of the cell surface protein in a drug-dependent manner.

Drug-inducible display of Notch and syn-Notch ligands: NS3 is also used to regulate the presentation of Notch and syn-Notch ligands on the surface of engineered mammalian cells. NS3 is integrated into cell-surface ligands such that they are retained on membrane surfaces (and thus able to activate Notch receptors) only in the presence of drug. Work described herein demonstrates that drug-dependent Notch signaling by cells expressing and NS3-fused version of the the Notch ligand DLL1 (DLL1-NS3).

Regulation of therapeutic cell activity: In addition to the further development of new drug-sensitive TFs and signaling proteins, dCNV and NS3-containing Notch/syn-Notch ligands are applied to regulate the therapeutic properties of engineered T cells, such as such as target-specificity, cell-killing ability, and self-destruction. A key aspect of the proposed approach is that the several FDA approved NS3 inhibitors are already used in the clinic to treat HCV. Thus, these systems are used to modulate therapeutic cells in vivo using molecules that are orally available and already known to be safe.

Materials and Methods

DNA Constructs

Plasmid DNA and detailed sequence information for expression vectors encoding Gal4DB-NS3-Gal4TA, Gal4DB-NS3-VP64, Gal4DB-NS3-VP64-p65, rTetR-NS3-VP64-p65, myr-palm-NS3-Gal4min, dCas9-NS3-NLS/VPR, and SNAP-dCas9-NS3-NLS/VPR can be obtained via AddGene. Standard cloning procedures were used in the generation of all DNA constructs. The pEV-UAS-H2B-citrine reporter plasmid was a gift from Michael Elowitz (Caltech), the TRE-mTagBFP reporter plasmid was a gift from Wilson Wong (Boston University), the 5×GAL4-TATA-luciferase reporter plasmid (Addgene #46756) was a gift from Richard Maurer (Oregon Health Sciences University), the Tet-inducible mCherry reporter (Addgene #64128) and sgRNA1_Tet-inducible Luciferase plasmids (Addgene #64128) were gifts from Moritoshi Sato (University of Tokyo).

NS3 Inhibitors

Asunaprevir, boceprevir, danoprevir, MK-5172 (a.k.a., grazoprevir), and simeprevir were from MedChemExpress. Telaprevir was from Selleck Chemicals. BILN-2061 was a gift from Roger Tsien and Stephen Adams (UC San Diego). Concentrated NS3 inhibitor stocks were dissolved in DMSO at concentrations between 3-10 mM and diluted into cell culture media at the indicated working concentrations.

Mammalian Cell Culture

All mammalian cell lines were cultured in a humidified incubator maintained at 37° C. with 5% CO2. HEK 293FT cells (ThermoFisher) were cultured in Dulbecco's modified Eagle medium (DMEM) containing with 10% FBS and supplemented with nonessential amino acids (Life Technologies), Glutamax (Life Technologies), and G418 (500 μg/mL, Invivogen). HeLa cells were obtained from ATCC and were cultured in DMEM containing 10% FBS and supplemented with Glutamax and penicillin/streptomycin. Stable cell lines based on CHO T-REx (ThermoFisher) were were maintained in DMEM containing 10% FBS and supplemented with nonessential amino acids and glutamax.

DNA Transfections

DNA transfections were performed using Lipofectamine 3000 Reagent (ThermoFisher) according to manufacturer's instructions. For imaging experiments, cells were seeded in dishes or well plates containing coverslip bottoms either coated with bovine plasma fibronectin (Product #F1141, Sigma-Aldrich) or treated for cell-adherence by the manufacturer (poly-D-lysine by MatTek, or ibiTreat by Ibidi).

Stable Cell Line Generation

Stable cell lines were generated from previously reported CHO-K1 T-REx cells containing stably integrated Gal4- and Notch-dependent reporter constructs (UAS H2B-Citrine and 12×CSL H2B-Citrine, respectively), which were gifts from Michael Elowitz (Caltech). Briefly, cells were transfected with linearized DNAs encoding the engineered protein of interest as well as antibiotic resistance gene for mammalian selection. Transfections were performed in 24-well plates containing 160,000 cells per well seeded the approximately 24 hours prior to transfection. At 48 hours post-transfection, the cells were transferred to 6-well plates and exposed to antibiotic selection using hygromycin (500 μg/mL). Upon elimination of non-transfected control cells (typically after 10 days of culture in the presence of antibiotic), surviving cells were transferred into 96-well plates using a limited dilution procedure in order to isolate single clones.

Antibodies

The following primary antibodies were used: mouse anti-Cas9 (Santa Cruz Biotechnology, sc-517386, 1:500 dilution for western blotting, 1:50 for immunostaining), mouse anti-human CD184 (CXCR-4) APC conjugate (BioLegend, 306510, 1:200 dilution for flow cytometry), polyclonal sheep anti-mouse/rat Dll1 (R&D Systems, AF3970, 1:50 dilution for immunostaining), rabbit anti-Histone H2B (Cell Signalling, 12364, 1:1,000 dilution for western blotting), mouse anti-HA-HRP (Santa-Cruz, sc-7392, 1:1,000 dilution for blotting), rabbit anti-GAPDH (Sigma-Aldrich, G9545, 1:3,000 dilution for western blotting), and polyclonal rabbit anti-Gal4 (Santa Cruz Biotechnology, sc-577, diluted 1:500 for western blotting, 1:200 for immunostaining). The following secondary antibodies were used: goat anti-human AlexaFluor647 conjugate (ThermoFisher, A-21445, 1:1,000 dilution), donkey anti-sheep AlexaFluor647 conjugate (ThermoFisher, A-21448, 1:1,000 dilution), goat anti-rabbit CF647 conjugate (Sigma-Aldrich, SAB4600184, 1:300 dilution), anti-mouse HRP conjugate (Cell Signalling, 7076, 1:3,000 dilution), and anti-rabbit HRP conjugate (Bio-Rad, 170-6515, 1:3,000 dilution).

Preparation of Cell Lysates for Immunoblotting

Cell lysates used in immunoblotting analyses were prepared by direct lysis of drug-treated and untreated cells in 1×LDS-PAGE loading buffer (ThermoFisher) following removal of cell culture media. Such procedure was applied in order to immediately denature proteins upon lysis, thus preventing undesired NS3 cis-cleavage in cell lysates. Viscous solutions were formed upon addition of the lysis reagent, which were clarified through sonication followed by centrifugation. The lysates were subsequently analyzed by standard immunoblotting procedures and probed using the antibodies listed above at the indicated dilutions. Detection of the labeled antigens was carried by chemiluminescence via the SuperSignal West Pico PLUS Chemiluminescent Substrate (Pierce).

Immunofluorescence Staining of Fixed Cells

Cells were rinsed with PBS prior to fixation with formaldehyde (4% v/v, diluted into PBS from fresh vials containing 16% solutions purchased from ThermoFisher). Cells were fixed for 10 minutes at room temperature, followed by rinsing with PBS (three times) to remove residual fixative. When necessary, cells were then permeabilized with Triton-X 100 (0.2%, v/v, in PBS) for 10 minutes, following by rinsing with PBS. Cells were blocked with BSA solution (5%, v/v in PBS) for approximately 30 minutes at room temperature prior to staining with primary antibody solution (typically in PBS, or in the appropriate solution as suggested by the antibody supplier) at the dilutions indicated above for 1 hour at room temperature. Cells were stained with secondary antibody solution (in PBS at the solutions indicated above) for 1 hour at room temperature before imaging.

Time-Dependent Dye Labeling of SNAP-Tagged Proteins

HeLa cells were transfected with DNA encoding SNAP-dCas9-NS3-NLS/VPR as described above herein. Approximately 24 hours later, cells were labeled with the red fluorescent dye SNAP-Cell TMR STAR (New England Biolabs) in complete culture media according to the manufacturer's protocol. The dye was removed by gentle aspiration of the media, followed by rinsing with pre-warmed complete media (three times) to remove residual dye. Cells were subsequently incubated in culture media containing 3 μM BILN-2061. After 8 hours, cells were then labeled with the green fluorescent SNAP-Cell Fluorescein (New England Biolabs). Cells were fixed with 4% formaldehyde prior to imaging.

Luciferase Assay

CHO-K1 cells stably expressing Gal4DB-NS3-VP64 were transfected with DNA encoding a UAS regulated firefly luciferase reporter construct (5×GAL4-TATA-luciferase). A constitutively transcribed NanoLuciferase construct (pNL1.1.TK[Nluc/TK], Promega) was used as a co-transfection control. Approximately 16 hours after transfection, cells were treated with either BILN-2061, or Grazoprevir (both at 3 μM). Following a 12 hour period of drug treatment, a time-series was initiated during which the drug-containing media was removed from individual wells and replaced with drug-free media over the course of a 48 hour period. At the end of the series (approximately 56 hours after the initial drug exposure, and 72 hours after transfection), the amount of luciferase and NanoLuciferase present in cells was quantified using the Nano-Glo Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. The CHO-K1 cell line used in these analyses also contained a stably integrated UAS H2B-Citrine reporter construct, and fluorescence imaging confirmed the activation of the Gal4-dependent H2B-citrine gene in all drug treated-wells.

Image Acquisition and Analysis

Cells were imaged by epifluorescence microscopy in imaging-compatible vessels containing glass coverslip bottoms (MatTek) or optically clear plastic bottoms (ibidi). During imaging, cells were maintained in PBS, standard culture media, or FluoroBrite DMEM (ThermoFisher). For time-lapse analyses, cells were imaged in culture media supplemented with 30 mM HEPES diluted from a 1 M stock (pH 7.2-7.5, ThermoFisher) and maintained at 37° C. in a heated imaging chamber throughout the duration of the analysis (typically 24 hours). Images were acquired using the ZEN imaging software (Zeiss). Image files were processed using the ImageJ-based image analysis package Fiji. The images were contrasted uniformly across experiments, and where applicable, pixel intensity profiles were plotted using the plot profile tool in Fiji. For the time-lapse analyses, movies were created using the ZEN imaging software.

Flow Cytometry

Cells analyzed by flow cytometry were gated for living cells by scatter detection. The geometric mean measured reporter fluorescence levels were reported in arbitrary fluorescence units (AFU). Reporter activation analyses were performed using stable single-clones, or cells transiently transfected with DNA encoding the analyzed TF (as indicated in the figure captions). For analyses carried out using transiently expressing cells, plasmid DNA encoding a constitutively expressed fluorescent protein marker was co-delivered at the time of transfection and used to identify positively transfected cells populations (see Supplementary FIG. 12). Briefly, transfected cells were were gated to the top 1% of marker fluorescence of non-transfected control cells under the same condition. Transient expression experiments carried out using the “turn-on” TFs (shown in FIGS. 1d and 1 i, and Supplementary FIG. 3) were gated via detection of an mCherry marker that was expressed via an IRES sequence on the TF-encoding plasmid. Transfected cells were incubated for 24-48 h after transfection either in the presence or absence of the indicated NS3 inhibitors before being analysed using an Attune N×T flow cytometer (ThermoFisher). For the analyses in which rTetR-NS3-VP64-p65 and TMD-NS3-Gal4min were combined, 125,000 HEK 293FT cells were transfected with 25 ng of DNA (per −125,000 cells) with DNA mixtures containing a 3:3:2:2 molar ratio of rTetR-NS3-VP64-p65 to TMD-NS3-Gal4 to TRE mTagBFP to UAS H2B Citrine (as approximated by DNA size).

For expression analyses carried out using dCas9-based TFs, HEK 293FT cells were seeded in 48-well plates and transfected at a density of −80,000 cells per well. A total 250 ng of DNA was delivered per well in each experiment; sgRNA- and dCas9-encoding constructs were transfected at a 1:1 molar ratio as approximated based on DNA size. The constructs encoding the sgRNAs targeting human CXCR4 promoter were acquired from AddGene and were previously reported (“sgC2” and “sgC3”). Expression of the chemokine receptor was analysed via flow cytometry using a fluorescently-conjugated CXCR-4 antibody; geometric means were recorded.

Statistics and Reproducibility

All flow cytometry and luminescence assay data were collected using 3 biologically independent samples. For fluorescence imaging analyses, ≥3 images per condition were recorded (encompassing hundreds of cells), and representative images are displayed in the figures. For immunoblotting, analyses were repeated ≥3 times and a representative blots were chosen for display.

Bulky Ectodomain Mechanoreceptor Embodiment

Gamma secretase is a membrane protein complex involved in biological functions such as Notch and amyloid precursor protein (APP) processing. Its proteolytic subunit, presenilin, acts by catalyzing the cleavage of intramembrane alpha helices, and in turn allows the release of both the extracellular domain (important in APP pathology) and the intracellular domain (important in Notch developmental biology). The gamma secretase extracellular subunit, nicastrin, has been shown to regulate this process through steric hindrance. Gamma secretase substrates with bulky extracellular domains are resistant to proteolysis, and the regulated shedding of this bulky ectodomain is a key pathway in Notch processing.

Shown here in FIG. 30 are the results using a novel mechanoreceptor with a fluorescent protein as its bulky ectodomain. Green fluorescent protein has been shown to unfold at approximately 100 pN (Dietz 2004), and unfolding of this domain would reduce steric hindrance and in turn allow the release of an intracellular transcription factor through gamma secretase processing.

Cells transiently transfected with DNA encoding this receptor display increased activation when plated on wells coated with an antibody that binds it. However, this increase in activation only occurred when the antibodies are tethered, and thus are able to apply force to the receptors: soluble antibodies at similar concentrations did not increase activation. Additionally, this process is supported to be gamma secretase dependent, as addition of a gamma secretase inhibitor diminished cell activation.

Wells from an untreated 96 well plate were coated overnight (20 hrs) with 0.005% gelatin and with or without 1 μg of an anti-myc antibody. They were then washed 3 times with 2004 PBS, and reporter cells that had been transfected with a plasmid encoding this protein were plated with media (10% FBS No antibiotics) with or without DAPT, a gamma secretase inhibitor (GS inhibitor). 2 hours later, anti-myc antibody was added to wells, and the cells were cultured overnight (20 hrs) at 37 degrees Celsius. All results are from flow cytometry, gated for live GFP+ cells. 3 samples for each condition.

REFERENCES

-   1. Lienert, F., Lohmueller, J. J., Garg, A., & Silver, P. A. (2014).     Synthetic biology in mammalian cells: next generation research tools     and therapeutics. Nature Reviews Molecular Cell Biology, 15, 95107. -   2. Failla, C., Tomei, L., & De Francesco, R. (1994). Both NS3 and     NS4A are required for proteolytic processing of hepatitis C virus     nonstructural proteins. Journal of Virology, 68(6), 3753-3760. -   3. Lin, M. Z., Glenn, J. S., & Tsien, R. Y. (2008). A     drug-controllable tag for visualizing newly synthesized proteins in     cells and whole animals. Proceedings of the National Academy of     Sciences,105(22), 7744-7749 -   4. Butko, M. T., Yang, J., Geng, Y., Kim, H. J., Jeon, N. L., Shu,     X., Mackey, M. R., Ellisman, M. H., Tsien, R. Y. & Lin, M. Z.     (2012). Fluorescent and photo-oxidizing TimeSTAMP tags track protein     fates in light and electron microscopy. Nature Neuroscience, 15(12),     1742-1751. -   5. Palida, S. F., Butko, M. T., Ngo, J. T., Mackey, M. R., Gross, L.     A., Ellisman, M. H., & Tsien, R. Y. (2015). PKMζ, but not PKCλ, is     rapidly synthesized and degraded at the neuronal synapse. Journal of     Neuroscience, 35(20), 7736-7749. -   6. Chung, H. K., Jacobs, C. L., Huo, Y., Yang, J., Krumm, S. A.,     Plemper, R. K., Tsien, R. Y. & Lin, M. Z. (2015). Tunable and     reversible drug control of protein production via a self-excising     degron. Nature Chemical Biology, 11(9), 713-720. -   7. Chavez, A., Scheiman, J., Vora, S., Pruitt, B. W. , Tuttle, M,     Iyer, E., Lin, S., Kiani, S, Guzman, C., Wiegand, D. J.,     Ter-Ovanesyan, D., J Braff, J. L., Davidsohn, E., Housden, B. E.,     Perrimon, N., Weiss, R., Aach, J., Collins, J. J., & Church, G. M.     Highly efficient Cas9-mediated transcriptional programming. Nature     Methods, 12(4), 326-328. -   8. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., &     Bujard, H. (1995). Transcriptional activation by tetracyclines in     mammalian cells. Science, 268(5218), 1766-1768. -   9. Zacharias, D. A., Violin, J. D., Newton, A. C., & Tsien, R. Y.     (2002). Partitioning of lipid-modified monomeric GFPs into membrane     microdomains of live cells. Science, 296(5569), 913-916. -   10. Perez-Pinera, Pablo, et al. RNA-guided gene activation by     CRISPR-Cas9-based transcription factors. Nature Methods 10.10     (2013): 973-976. -   11. Maeder, Morgan L., et al. CRISPR RNA-guided activation of     endogenous human genes. Nature Methods 10.10 (2013): 977-979. -   12. Qi , L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A.,     Weissman, J. S., Arkin, A. P., & Lim, W. A. Repurposing CRISPR as an     RNA-guided platform for sequence-specific control of gene     expression. Cell 152.5 (2013): 1173-1183. -   13. Zalatan, J. G., Lee, M. E., Almeida, R., Gilbert, L. A.,     Whitehead, E. H., La Russa, M., Tsai, J. C., Weissman, J. S.,     Dueber, J. E., Qi, L. S. & Lim, W. A. (2015). Engineering complex     synthetic transcriptional programs with CRISPR RNA scaffolds. Cell,     160(1), 339-350. -   14. Komor, Alexis C., Ahmed H. Badran, and David R. Liu.     CRISPR-based technologies for the manipulation of eukaryotic     genomes. Cell 168.1-2 (2017): 20-36. -   15. Gordon, W. R., Zimmerman, B., He, L., Miles, L. J., Huang, J.,     Tiyanont, K., McArthur, D. G., Aster, J. C., Perrimon, N.,     Loparo, J. J., & Blacklow, S. C (2015). Mechanical allostery:     evidence for a force requirement in the proteolytic activation of     Notch. Developmental Cell, 33(6), 729-736. -   16. Morsut, L., Roybal, K. T., Xiong, X., Gordley, R. M., Coyle, S.     M., Thomson, M., & Lim, W. A. (2016). Engineering customized cell     sensing and response behaviors using synthetic notch receptors.     Cell, 164(4), 780-791. -   17. Roybal, K. T., Rupp, L. J., Morsut, L., Walker, W. J.,     McNally, K. A., Park, J. S., & Lim, W. A. (2016). Precision tumor     recognition by T cells with combinatorial antigen-sensing circuits.     Cell, 164(4), 770-779. -   18. Varnum-Finney, B., Wu, L., Yu, M., Brashem-Stein, C., Staats,     S., Flowers, D., Griffin, J. D., & Bernstein, I. D. (2000).     Immobilization of Notch ligand, Delta-1, is required for induction     of notch signaling. Journal of Cell Science, 113(23), 4313-4318. -   19. Sprinzak, D., Lakhanpal, A., LeBon, L., Santat, L. A.,     Fontes, M. E., Anderson, G. A., Garcia-Ojalvo, J. & Elowitz, M. B.     (2010). Cis interactions between notch and delta generate mutually     exclusive signaling states. Nature, 465(7294), 86.

Example 3

Reduction of SynNotch Leakiness by Incorporation of the Juxtamembrane LWF Motif.

SynNotch receptors are designed around a preserved Notch regulatory “core,” the minimal protein unit identified as necessary for maintaining the natural Notch signaling mechanism. Specifically, SynNotch cores retain the Negative Regulatory Region (NRR) and Transmembrane Domain (TMD) of natural Notch. Although SynNotch receptors successfully mimic many key Notch signaling characteristics, they fail to recapitulate some regulatory aspects of Notch signaling; natural Notch signaling is tightly regulated, but SynNotch signaling is often observed as noisy or “leaky,” with some SynNotch constructs having significant background activation even in the absence of stimulus.

The leakiness of SynNotch receptors may be due the introduction of synthetic protein modules, the absence of natural Notch components, or likely some combination thereof. For example, it has been observed that addition of more powerful transcriptional activators (VPR, as opposed to VP64) leads to an extreme increase in background noise and renders the receptors near inviable.

Pursuing the hypothesis that the SynNotch regulatory core may be missing an element of the natural receptor important for regulating signaling dynamics, Notch's “LWF motif” was identified as a candidate for reintroduction into SynNotch. The LWF motif, absent in SynNotch, is a hydrophobic stretch of amino acids juxtamembrane to the TMD's C-terminus that was recently modeled to briefly reenter the cytosoloic face of the cell membrane [1] (FIG. 31A). The TMD of Notch contains the S3 site for cleavage by γ-secretase, the final proteolytic event necessary for receptor activation. Dysregulated cleavage by γ-secretase is a likely cause of SynNotch leakiness, as indicated by the ability of DAPT (γ-secretase inhibitor) to suppress leaky receptor activation. Due to the LWF motif's proximity with the TMD and S3, as well as modeled interactions with the cell membrane, without wishing to be bound by a particular theory, it was hypothesized that inclusion of the motif could restore some natural regulation of receptor activity. Because the modeled LWF motif structure was not published until after SynNotch, it was likely not considered in the original SynNotch core design.

Data herein provides preliminary evidence that inclusion of the LWF motif reduces background activation in SynNotch receptors. In initial experiments, the original SynNotch core design (SN) was compare with a SynNotch core containing the LWF motif (SN-LWF) (FIG. 31A). Each receptor core was test with two different transcription factor intracellular domains (ICD's): Gal4 fused to VP64 (“VP64,” a modest transcriptional activator commonly used in SynNotch) and Gal4 fused to VPR (“VPR,” a strong transcriptional activator, whose strength is penalized by high background activation in SynNotch constructs). For VP64 ICD's, it was found that both SN-VP64 and SN-LWF-VP64 have low background activation and activate to the same extent when stimulated, as expected for the modest transcriptional activator (FIG. 31B). For the stronger VPR ICD, however, it was found that SN-VPR is significantly leakier than SN-LWF-VPR, while both activate to comparable extents (FIG. 31C). These data indicate that inclusion of the LWF motif in SynNotch may permit the use of valuable transcriptional effectors that are not viable in leaky settings. For example, if tightly regulated like natural Notch signaling, potent domains such as VPR could allow more rapid transcriptional outputs in therapeutic settings, or DNA editors such as Cre and Cas9 could be used without risk of erroneous gene editing.

REFERENCE

-   [1] Deatherage et al. “Structural and biochemical differences     between the Notch and the amyloid precursor protein transmembrane     domains.” Science Advances, 2017.

Example 4

Previously, a design strategy and preliminary data for SynNotch receptors with programmable force-activation thresholds was introduced. Natural and synthetic Notch receptors contain a Negative Regulatory Region (NRR), which is an auto-inhibitory domain that opens in response to tensile forces greater than ˜5 pN. This mechanical opening of the NRR is necessary for downstream transcriptional activity of the receptor. In order to create Notch-based receptors with force-activation thresholds above 5 pN, an scFv targeting the NRR was fused to the N-terminus of the NRR itself. Receptors containing this scFv-NRR (“sNRR”) domain require greater tensile force for activation due to the additional protein interaction that must be disrupted to open the NRR. Furthermore, the precise amount of force required for receptor activation is determined by scFv binding affinity for the NRR and is thus tunable.

Provided herein are additional embodiments of the synthetic mechanoreceptors, their sNRR domains, and the potential applications of the invention described herein. In certain embodiments, the design criteria for building functional mechanoreceptors include, but are not limited to: they must be expressed at the cell surface (FIGS. 32A and 32B), they must activate in response to physiologically relevant and measurable forces (FIGS. 33A and 33B), and this force-activation threshold must be tunable (FIG. 34A-34E).

FIG. 35 shows that SynNotch receptors containing a sNRR domain expressed at the surface of HeLa cells similarly to receptors containing the WT NRR domain. Data presented in FIG. 35 indicate that the receptors can be stably expressed in HEK 293FT cells.

Previously herein, the use of a tension gauge tether (TGT) assay to quantify mechanostability in the engineered receptors described herein was introduced. FIGS. 33A and 33B show evidence for the increased mechanical strength of the sNRR domain described herein. While the WT NRR opens in response to ˜5 pN of tensile force, the sNRR domain has a tension tolerance closer to ˜50 pN.

Next, it was set out to tune the mechanical strength of sNRR and create a collection of receptors that activate in response definable and physiologically relevant forces. Because the unbinding force of an antibody-antigen pair correlates with its thermal dissociation rate, it was hypothesized that sNRR tension tolerance could be altered by mutating affinity of the scFv for the NRR. Previously presented data shows proof-of-concept evidence for this approach, using two model point mutations on the scFv, which was expressed as a separate transmembrane cis-inhibitor. In FIG. 34A-34E presents further evidence supporting this design strategy. A collection of mutated sNRR domains exhibiting a spectrum of mechanical strengths was generated. Notably, the impact of residue mutation on receptor strength followed predictable trends; more severe point mutations resulted in weaker receptors (Y49A weaker than Y49F, R99A weaker than R99K), and double point mutations were additively weaker than their constituents. Time-course microscopy of cells expressing mutated sNRR receptors further demonstrates the receptors' distinct mechanical characteristics, as well as their ability to discriminate between mechanical stimuli over time. Lastly, provided herein is a mathematical model that captures the observed temporal trends in receptor activation, simply by varying (1) the scFv-NRR dissociation rate vs. (2) the dissociation rate of the TGT double stranded DNA. The ability of such a simple two-parameter model to recapitulate our observed results indicates that mutating scFv-NRR affinity is indeed sufficient to create mechanically distinct receptors.

Importantly, the mechanoreceptors presented herein not only measure an applied force, but respond to and make a decision based off that force. In various embodiments, designer responses to force may be therapeutic in nature, or geared to study/recapitulate natural phenomenon in mechanobiology. For example, stem cells have been shown to differentiate down lineages dictated by their mechanical environment. Presented herein in FIG. 35, this natural process was mimic by using sNRR-based SynNotch to drive myogenic differentiation of fibroblast cells based on mechanical TGT stimulus.

Lastly, data presented herein show that the choice of NRR-binding scFv is nontrivial in the design of sNRR (FIG. 36). Three additional antibodies known to bind the NRR and inhibit Notch activation were incorporated into sNRR domains as scFv's. Although known to be effective as soluble antibodies, these scFv's offer little to no additional mechanical stability to the receptor. The original sNRR was designed to accommodate binding of the particular scFv in use (based off the crystal structure of the antibody-antigen complex), and incorporation of different scFv's would require additional engineering to optimally position them with respect to the NRR.

Sequences

Provided herein are sequences for use, in whole or in part, in the various embodiments of the compositions, methods, and systems described herein.

SEQ ID NO: 1 is an amino acid sequence encoding signal Sequence (SS).

(SEQ ID NO: 1) AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRR T

SEQ ID NO: 2 is an amino acid sequence encoding human Notch1 LBD.

(SEQ ID NO: 2) RGPRCSQPGETCLNGGKCEAANGTEACVCGGAFVGPRCQDPNPCLSTPCK NAGTCHVVDRRGVADYACSCALGFSGPLCLTPLDNACLTNPCRNGGTCDL LTLTEYKCRCPPGWSGKSCQQADPCASNPCANGGQCLPFEASYICHCPPS FHGPTCRQDVNECGQKPGLCRHGGTCHNEVGSYRCVCRATHTGPNCERPY VPCSPSPCQNGGTCRPTGDVTHECACLPGFTGQNCEENIDDCPGNNCKNG GACVDGVNTYNCRCPPEWTGQYCTEDVDECQLMPNACQNGGTCHNTHGGY NCVCVNGWTGEDCSENIDDCASAACFHGATCHDRVASFYCECPHGRTGLL CHLNDACISNPCNEGSNCDTNPVNGKAICTCPSGYTGPACSQDVDECSLG ANPCEHAGKCINTLGSFECQCLQGYTGPRCEIDVNECVSNPCQNDATCLD QIGEFQCICMPGYEGVHCEVNTDECASSPCLHNGRCLDKINEFQCECPTG FTGHLCQYDVDECASTPCKNGAKCLDGPNTYTCVCTEGYTGTHCEVDIDE CDPDPCHYGSCKDGVATFTCLCRPGYTGHHCETNINECSSQPCRHGGTCQ DRDNAYLCFCLKGTTGPNCEINLDDCASSPCDSGTCLDKIDGYECACEPG YTGSMCNINIDECAGNPCHNGGTCEDGINGFTCRCPEGYHDPTCLSEVNE CNSNPCVHGACRDSLNGYKCDCDPGWSGTNCDINNNECESNPCVNGGTCK DMTSGYVCTCREGFSGPNCQTNINECASNPCLNQGTCIDDVAGYKCNCLL PYTGATCEVVLAPCAPSPCRNGGECRQSEDYESFSCVCPTGWQAGQTCEV DINECVLSPCRHGASCQNTHGGYRCHCQAGYSGRNCETDIDDCRPNPCHN GGSCTDGINTAFCDCLPGFRGTFCEEDINECASDPCRNGANCTDCVDSYT CTCPAGFSGIHCENNTPDCTESSCFNGGTCVDGINSFTCLCPPGFTGSYC QHDVNECDSQPCLHGGTCQDGCGSYRCTCPQGYTGPNCQNLVHWCDSSPC KNGGKCWQTHTQYRCECPSGWTGLYCDVPSVSCEVAAQRQGVDVARLCQH GGLCVDAGNTHHCRCQAGYTGSYCEDLVDECSPSPCQNGATCTDYLGGYS CKCVAGYHGVNCSEEIDECLSHPCQNGGTCLDLPNTYKCSCPRGTQGVHC EINVDDCNPPVDPVSRSPKCFNNGTCVDQVGGYSCTCPPGFVGERCEGDV NECLSNPCDARGTQNCVQRVNDFHCECRAGHTGRRCESVINGCKGKPCKN GGTCAVASNTARGFICKCPAGFEGATCENDARTCGSLRCLNGGTCISGPR SPTCLCLGPFTGPECQFPASSPCLGGNPCYNQGTCEPTSESPFYRCLCPA KFNGLLCH

SEQ ID NO: 3 is an amino acid sequence encoding LaG17 anti-GFP nanobody.

(SEQ ID NO: 3) MADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFV AGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAV RTSGFFGSIPRTGTAFDYWGQGTQVTVS

SEQ ID NO: 4 is an amino acid sequence encoding scFv αFITC.

(SEQ ID NO: 4) QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAG LSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRS YDSSGYAGHFYSYMDVWGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSV SAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVP DRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL G

SEQ ID NO: 5 is an amino acid sequence encoding SNAP tag.

(SEQ ID NO: 5) MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPA AVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLW KLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQ GDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLG

SEQ ID NO: 6 is an amino acid sequence encoding FLAG tag.

DYKDDDDKG (SEQ ID NO: 6)

SEQ ID NO: 7 is an amino acid sequence encoding Myc tag.

EQKLISEEDL (SEQ ID NO: 7)

SEQ ID NO: 8 is an amino acid sequence encoding human NRR1.

(SEQ ID NO: 8) ILDYSFGGGAGRDIPPPLIEEACELPECQEDAGNKVCSLQCNNHACGWDG GDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQRAEG QCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVV VVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFPYYGREEEL RKHPIKRAAEGWAAPDALLGQVKASLLPGGSEGGRRRRELDPMDVRGSIV YLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLNIPYKIEAVQSETV EPPPPAQ

SEQ ID NO: 9 is an amino acid sequence encoding mouse NRR1.

(SEQ ID NO: 9) ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDG GDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEG QCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVL VVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEEL RKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCV QSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQ

SEQ ID NO: 10 is an amino acid sequence encoding mouse NRR 2.

(SEQ ID NO: 10) LYTAPPSTPPATCLSQYCADKARDGVCDEACNSHACQWDGGDCSLTMENPW ANCSSPLPCWDYINNQCDELCNTVECLFDNFECQGNSKTCKYDKYCADHFK DNHCDQGCNSEECGWDGLDCAADQPENLAEGTLVIVVLMPPEQLLQDARSF LRALGTLLHTNLRIKRDSQGELMVYPYYGEKSAAMKKQRMTRRSLPGEQEQ EVAGSKVFLEIDNRQCVQDSDHCFKNTDAAAALLASHAIQGTLSYPLVSVV SESLTPERTQ

SEQ ID NO: 11 is an amino acid sequence encoding NICD.

(SEQ ID NO: 11) QHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEW GDEDLETKKFRFEEPVVLPDLDDQTDHRQWTQQHLDAADLRMSAMAPTPPQ GEVDADCMDVNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPAVISDFIY QGASLHNQTDRTGETALHLAARYSRSDAAKRLLEASADANIQDNMGRTPLH AAVSADAQGVFQILIRNRATDLDARMHDGTTPLILAARLAVEGMLEDLINS HADVNAVDDLGKSALHWAAAVNNVDAAVVLLKNGANKDMQNNREETPLFLA AREGSYETAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLV RSPQLHGAPLGGTPTLSPPLCSPNGYLGSLKPGVQGKKVRKPSSKGLACGS KEAKDLKARRKKSQDGKGCLLDSSGMLSPVDSLESPHGYLSDVASPPLLPS PFQQSPSVPLNHLPGMPDTHLGIGHLNVAAKPEMAALGGGGRLAFETGPPR LSHLPVASGTSTVLGSSSGGALNFTVGGSTSLNGQCEWLSRLQSGMVPNQY NPLRGSVAPGPLSTQAPSLQHGMVGPLHSSLAASALSQMMSYQGLPSTRLA TQPHLVQTQQVQPQNLQMQQQNLQPANIQQQQSLQPPPPPPQPHLGVSSAA SGHLGRSFLSGEPSQADVQPLGPSSLAVHTILPQESPALPTSLPSSLVPPV TAAQFLTPPSQHSYSSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHS NVSDWSEGVSSPPTSMQSQIARIPEAFK

SEQ ID NO: 12 is an amino acid sequence encoding Gal4DBD-VP64.

(SEQ ID NO: 12) MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTR AHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNV NKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGS GGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDMLGS

SEQ ID NO: 13 is an amino acid sequence encoding human Notch1 TM.

(SEQ ID NO: 13) LHFMYVAAAAFVLLFFVGCGVLLSRKRRR

SEQ ID NO: 14 is an amino acid sequence encoding mouse Notch1 TM.

(SEQ ID NO: 14) LHLMYVAAAAFVLLFFVGCGVLLSRKRRR

SEQ ID NO: 15 is an amino acid sequence encoding Ab2; wherein in SEQ ID NO: 15, bold text depicts the VH domain, underlined text depicts the linker domain, and double underlined text depicts the VL domain).

(SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTV SSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

The amino acid sequences of SEQ ID NO:16-24, depict amino acid changes from SEQ ID NO:15

SEQ ID NO: 16 is an amino acid sequence encoding Ab2-VH N55A. The VH N55 mutation is depicted in bold, underlined text.

(SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPP A RSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 17 is an amino acid sequence encoding Ab2-VH R99K. The VH R55 mutation depicted in bold, underlined text.

(SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF K WVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 18 is an amino acid sequence encoding Ab2-VH R99A. The VH R99 mutation is depicted in bold, underlined text.

(SEQ ID NO: 18) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF A WVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 19 is an amino acid sequence encoding Ab2-VH C22S/C92A. The VH C22 and C92 mutations are depicted in bold, underlined text.

(SEQ ID NO: 19) EVQLVESGGGLVQPGGSLRLSSAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY A ARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 20 is an amino acid sequence encoding Ab2-VL S30A. The VL S30 mutation is depicted in bold, underlined text.

(SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDV A TAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK 

SEQ ID NO: 21 is an amino acid sequence encoding Ab2-VL Y49A. The VL Y49 mutation is depicted in bold, underlined text.

(SEQ ID NO: 21) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLI A SASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK 

SEQ ID NO: 22 is an amino acid sequence encoding Ab2-VL Y55A. The VL Y55 mutation is depicted in bold, underlined text)

(SEQ ID NO: 22) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFL A SGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIK

SEQ ID NO: 23 is an amino acid sequence encoding Ab2-VL F91A. The VL F91 mutation is depicted in bold, underlined text.

(SEQ ID NO: 23) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ A YTTPSTFGQGTKVEIK 

SEQ ID NO: 24 is an amino acid sequence encoding Ab2-VL Y92A. The VL Y92 mutation shown in bold, underlined text.

(SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARI NPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGF RWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVG DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQF A TTPSTFGQGTKVEIK

SEQ ID NO: 25 is an amino acid sequence encoding E6.

(SEQ ID NO: 25) QVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGLEWVAG LSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRS YDSSGYAGHFYSYMDVWGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSV SAAPGQKVTISCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVP DRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL G 

SEQ ID NO: 26 is an amino acid sequence encoding WC629.

(SEQ ID NO: 26) EVQLVQSGAEVKKPGSSVKVSCKASGGTLSSYTVSWLRQAPGQGLEWMGR IIPILDRANYAQKFQGRVTITADKSTSTAYMELNSLRSDDTAVYYCARSI GAAGDGVWFDPWGQGTMVTVSSGGSSRSSSSGGGGSGGGGQAVLTQPSSV SGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIFDNKNRPSGV PDRFSGSNSGTSASLAITGLQAEDEAEYYCQSYDNNLSGRVFGGGTKLTV

SEQ ID NO: 27 is an amino acid sequence encoding WC75.

(SEQ ID NO: 27) LVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSSISWHSRTIAY ADSVKGRFSISRDNAKNSLYLQMNSLRPEDTAVYYCAKASYLSTSSSLDY WGRGTLVTVSSGGSSRSSSSGGGGSGGGGQSVLTQPGSVSGSPGQSITIS CTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGN TASLTISGLQAEDEADYYCSSYTTRSTRVFGGGTKLTVL

SEQ ID NO: 28 is an amino acid sequence encoding D3.

(SEQ ID NO: 28) EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYGMSWVRQAPGKGLEWVSY IYPYSGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARHS GYYRISSAMDVWGQGTLVTVSAGGSSRSSSSGGGGSGGGGDIQMTQSPSS LSASVGDRVTITCRASQNIKRFLAWYQQKPGKAPKLLIYGASTRESGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRSPHTFGQGTKVEIKRGG

SEQ ID NO: 29 is an amino acid sequence encoding B6.

(SEQ ID NO: 29) AQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMG WMNAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARD RVPTIPAYRIDYWGQGTLVTVSSLEGGGGSGGGGSGGGASDIQMTQSPSS VSASVGDRVTITCRASQGISSWLAWYQQKPGKAPRLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKLEIKR

SEQ ID NO: 30 is an amino acid sequence encoding B9.

(SEQ ID NO: 30) AQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMG WINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARG PRSYGAGGMDVWGQGTLVTVSSLEGGGGSGGGGSGGGASDIQMTQSPSSV SASVGDRVTITCRASQGISSWLAWYQQKPGKAPKFLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKR

SEQ ID NO: 31 is an amino acid sequence encoding Platelet Derived Growth Factor Receptor (PDGFR) TM domain.

(SEQ ID NO: 31) AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRR T

SEQ ID NO: 32 is an amino acid sequence encoding NS3. In SEQ IC NO:32, the bold, underlined text depicts the N- and C-terminal cis-cleavage sites and AU1 tag.

(SEQ ID NO: 32) EDVVCCHSIYGKKKGDI DTYRYI GSSGTGCVVIVGRIVLSGSGTSAPITA YAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAV YHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLY LVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFR AAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDRE VLYQEFDEMEECSQH

SEQ ID NO: 33 is an amino acid sequence encoding AU1 tag.

(SEQ ID NO: 33) DTYRYI

SEQ ID NO: 34 is an amino acid sequence encoding rat Dll1 SS-ECD.

(SEQ ID NO: 34) MGRRSALALAVVSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGSG PPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTAVLGVDSFSLPDGAGID PAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLTTQR HLTVGEEWSQDLHSSGRTDLRYSYRFVCDEHYYGEGCSVFCRPRDDAFGH FTCGERGEKMCDPGWKGQYCTDPICLPGCDDQHGYCDKPGECKCRVGWQG RYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCRNG ATCTNTGQGSYTCSCRPGYTGANCELEVDECAPSPCRNGGSCTDLEDSYS CTCPPGFYGKVCELSAMTCADGPCFNGGRCSDNPDGGYTCHCPAGFSGFN CEKKIDLCSSSPCSNGAKCVDLGNSYLCRCQTGFSGRYCEDNVDDCASSP CANGGTCRDSVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHQRGQ RYMCECAQGYGGANCQFLLPEPPPDLIVAAQGGSFPW

SEQ ID NO: 35 is an amino acid sequence encoding rat Dll1 TM domain and ICD.

(SEQ ID NO: 35) VAVCAGVVLVLLLLLGCAAVVVCVRLKLQKHQPPPDPCGGETETMNNLAN CQREKDVSVSIIGATQIKNTNKKADFHGDHGADKSSFKARYPTVDYNLIR DLKGDEATVRDAHSKRDTKCQSQGSVGEEKSTSTLRGGEVPDRKRPESVY STSKDTKYQSVYVLSAEKDECVIATEV

SEQ ID NO: 36 is an amino acid sequence encoding human Dll4 SS-ECD.

(SEQ ID NO: 36) MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPC EPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGR NPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLA VGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVC QPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLC NECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATC SNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLC PPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCE KKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCA HGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLS TDTFVCNCPYGFVGSRCEFPVGLPPSFPW

SEQ ID NO: 37 is an amino acid sequence encoding human Dll4 TM domain and ICD.

(SEQ ID NO: 37) VAVSLGVGLAVLLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNL IPAAQLKNTNQKKELEVDCGLDKSNCGKQQNHTLDYNLAPGPLGRGTMPG KFPHSDKSLGEKAPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNEC VIATEV

SEQ ID NO: 38 is an amino acid sequence encoding mouse Dll4 SS-ECD.

(SEQ ID NO: 38) MTPASRSACRWALLLLAVLWPQQRAAGSGIFQLRLQEFVNQRGMLANGQS CEPGCRTFFRICLKHFQATFSEGPCTFGNVSTPVLGTNSFVVRDKNSGSG RNPLQLPFNFTWPGTFSLNIQAWHTPGDDLRPETSPGNSLISQIIIQGSL AVGKIWRTDEQNDTLTRLSYSYRVICSDNYYGESCSRLCKKRDDHFGHYE CQPDGSLSCLPGWTGKYCDQPICLSGCHEQNGYCSKPDECICRPGWQGRL CNECIPHNGCRHGTCSIPWQCACDEGWGGLFCDQDLNYCTHHSPCKNGST CSNSGPKGYTCTCLPGYTGEHCELGLSKCASNPCRNGGSCKDQENSYHCL CPPGYYGQHCEHSTLTCADSPCFNGGSCRERNQGSSYACECPPNFTGSNC EKKVDRCTSNPCANGGQCQNRGPSRTCRCRPGFTGTHCELHISDCARSPC AHGGTCHDLENGPVCTCPAGFSGRRCEVRITHDACASGPCFNGATCYTGL SPNNFVCNCPYGFVGSRCEFPVGLPPSFPWVA

SEQ ID NO: 39 is an amino acid sequence encoding mouse Dll4 TM domain and ICD.

(SEQ ID NO: 39) VSLGVGLVVLLVLLVMVVVAVRQLRLRRPDDESREAMNNLSDFQKDNLIP AAQLKNTNQKKELEVDCGLDKSNCGKLQNHTLDYNLAPGLLGRGGMPGKY PHSDKSLGEKVPLRLHSEKPECRISAICSPRDSMYQSVCLISEERNECVI ATEV

SEQ ID NO: 40 is an amino acid sequence encoding mCherry.

(SEQ ID NO: 40) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKW ERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG WEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPG AYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

SEQ ID NO: 41 is an amino acid sequence encoding SS for cis-inhibitors.

(SEQ ID NO: 41) METDTLLLWVLLLWVPGSTGDGGGG

SEQ ID NO: 42 is an amino acid sequence encoding an exemplary scFv (depicted in bold text) is expressed within a SynNotch as an “LNR4” domain. The underlined text depicts the linker, the double underlined, italic text depicts the linker with myc tag.

(SEQ ID NO: 42) GGGGSTGDGGGG EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWIHWV RQAPGKGLEWVARINPPNRSNQYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGSSRSSSSGGGGSGG GGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLL IYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFYTTPS TFGQGTKVEIK

SEQ ID NO: 43 is an amino acid sequence encoding an exemplary full construct for a mouse-based SynNotch with myc-tagged LBD that can bind FITC or GFP, an Ab2 LNR4, and a Gal4-VP64 ICD. SEQ ID NO: 43 comprises, from N to C, a Signal Sequence; LBD (which can comprise multiple domains to bind different ligands; depicted in bold text); Notch Core (which comprises a Notch1 NRR, and an optional LNR4 domain expressed N-terminal to the NRR, depicted in underlined text); TM domain (depicted in italicized text); and an ICD (depicted in bold, underlined text).

(SEQ ID NO: 43) MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLVESGGNLVQPGGSL RLSCAASGFTFGSFSMSWVRQAPGGGLEWVAGLSARSSLTHYADSVKGR FTISRDNAKNSVYLQMNSLRVEDTAVYYCARRSYDSSGYAGHFYSYMDV WGQGTLVTVSGGGGSGGGGSGGGGSSVLTQPSSVSAAPGQKVTISCSGS TSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASL DISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGGGGSMADVQLVE SGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSA GSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFF GSIPRTGTAFDYWGQGTQVTV SGGGGSTGDGGGGEVQLVESGGGLVQPG GSLRLSCAASGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSV KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGT LVTVSSGGSSRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRA SQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFYTTPSTFGQGTKVEIKGGGSEQKLISEEDLGG GSILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACG WDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQ LTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAA GTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYY GHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLE IDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEP PLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR MKLLSSIEQACDICR LKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERL EQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLAS VETDMPLTLRQHRISATSSSEESSNKG Q R Q LTVSAAAGGSGGSGGSDAL DDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS

SEQ ID NO: 44 is an amino acid sequence encoding an exemplary scFv expressed as a separate transmembrane cis-inhibitor of Notch. SEQ ID NO: 44 comprises, from N to C,Signal Sequence; scFV (depicted in bold text); Myc Tag and TM domain (depicted in italicized text); and mCherry (depicted in underlined text).

(SEQ ID NO: 44) METDTLLLWVLLLWVPGSTGDGGGGEVQLVESGGGLVQPGGSLRLSCAA SGFTFSSYWIHWVRQAPGKGLEWVARINPPNRSNQYADSVKGRFTISAD TSKNTAYLQMNSLRAEDTAVYYCARGSGFRWVMDYWGQGTLVTVSSGGS SRSSSSGGGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVA WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQFYTTPSTFGQGTKVEIK GGGSEQKLISEEDLGGGSAVGQDTQ EVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRRT MVSKG EEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASS ERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVN IKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

SEQ ID NO: 45 is an amino acid sequence encoding an exemplary full DLL1-NS3 construct (rat Dll1) with C-terminal mCherry. SEQ ID NO: 45 comprises, from N to C, Signal sequence, Dll1 ECD; T7 tag (depicted in bold text); NS3; HA Tag (depicted in italicized text); TM domain; Dll1 ICD; and mCherry (depicted in underlined text.)

(SEQ ID NO: 45) MGRRSALALAVVSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGS GPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTAVLGVDSFSLPDGAG IDPAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLT TQRHLTVGEEWSQDLHSSGRTDLRYSYRFVCDEHYYGEGCSVFCRPRDD AFGHFTCGERGEKMCDPGWKGQYCTDPICLPGCDDQHGYCDKPGECKCR VGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHH KPCRNGATCTNTGQGSYTCSCRPGYTGANCELEVDECAPSPCRNGGSCT DLEDSYSCTCPPGFYGKVCELSAMTCADGPCFNGGRCSDNPDGGYTCHC PAGFSGFNCEKKIDLCSSSPCSNGAKCVDLGNSYLCRCQTGFSGRYCED NVDDCASSPCANGGTCRDSVNDFSCTCPPGYTGRNCSAPVSRCEHAPCH NGATCHQRGQRYMCECAQGYGGANCQFLLPEPPPDLIVAAQGGSFPWSR ADMASMTGGQQMGSTEDVVCCHSIYGKKKGDIDTYRYIGSSGTGCVVIV GRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVST ATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPA PQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLK GSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFT DNSSPPAVTLTHPITKIDREVLYQEFDEMEECSQHYPYDVPDYAGASAV AVCAGVVLVLLLLLGCAAVVVCVRLKLQKHQPPPDPCGGETETMNNLAN CQREKDVSVSIIGATQIKNTNKKADFHGDHGADKSSFKARYPTVDYNLI RDLKGDEATVRDAHSKRDTKCQSQGSVGEEKSTSTLRGGEVPDRKRPES VYSTSKDTKYQSVYVLSAEKDECVIATEVVSKGEEDNMAIIKEFMRFKV HMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQF MYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQ DGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQ RLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVE QYERAEGRHSTGGMDELYKS

SEQ ID NO: 50 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(Gal4). DB_(Gal4) is in bolded text, TA_(Gal4) is in underlined text, NS4A is in italicized text, NS3 is in bolded, underlined text, and NS3 Cut Site is in bolded, italicized text.

(SEQ ID NO: 50) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGA AGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAA GAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTT ACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAAC AACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAA GATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTG CAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGG AGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAG CTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGT gggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGT CGACG

CGGCAAGAAGAAGGG TGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTG GTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCA TCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCAC CAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATC GTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTAT GCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAA GGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGC TGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCT CCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCG CCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCC TACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACG CCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGC GGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCG GTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAA TCACCAAAATCGATAGGGAGGTT

TATCCCTACGATGTGCCCGATTACGCTGG CGCGTCTGCATGCGCCAACTTTAATCAAAGTGGAAACATCGCGGACAGC TCACTCAGCTTTACCTTCACCAATAGCAGTAACGGGCCGAACCTCATAA CCACCCAGACCAACAGCCAGGCCTTGAGCCAGCCGATCGCCTCATCTAA CGTGCATGATAACTTTATGAACAACGAGATCACCGCGAGTAAGATAGAC GACGGGAACAACAGCAAGCCCCTTAGCCCAGGTTGGACGGACCAGACCG CCTACAACGCTTTCGGCATTACGACCGGCATGTTCAACACCACGACCAT GGACGATGTGTACAACTACCTGTTCGATGACGAAGACACACCGCCAAAC CCCAAAAAAGAA

SEQ ID NO: 51 is a nucleotide sequence encoding DB_(Gal4)-NS3-TAVP32. DBGal4 is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3 Cut Site is in bold/italics, NLS underlined, VP32 is in bold/italics/underlined

(SEQ ID NO: 51) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGA AGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAA GAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTT ACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAAC AACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAA GATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTG CAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGG AGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAG CTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGT gggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGT CGACG

CGGCAAGAAGAAGGG TGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTG GTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCA TCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCAC CAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATC GTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTAT GCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAA GGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGC TGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCT CCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCG CCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCC TACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACG CCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGC GGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCG GTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAA TCACCAAAATCGATAGGGAGGTT

TATCCCTACGATGTGCCC GATTACGCTGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGT CGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAA AAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCT

SEQ ID NO: 52 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(VP64). DB_(Gal4) is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3 Cut Site is in bold/italics, NLS is underlined, and VP64 is in bold/italics/underlined.

(SEQ ID NO: 52) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGA AGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAA GAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTT ACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAAC AACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAA GATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTG CAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGG AGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAG CTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGT gggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGT CGACG

CGGCAAGAAGAAGGG TGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTG GTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCA TCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCAC CAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATC GTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTAT GCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAA GGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGC TGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCT CCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCG CCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCC TACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACG CCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGC GGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCG GTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAA TCACCAAAATCGATAGGGAGGTT

TATCCCTACGATGTGCCCGATTACGCTGG CGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATC CGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCT ACAAAGAGGCCAGCGGTTCCGGACGGGCT

SEQ ID NO: 53 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(VP64-p65). DB_(Gal4) is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3 Cut Site is in bold/italics, NLS is underlined, VP64 is in bold/italics/underlined, P65 is double underlined.

(SEQ ID NO: 53) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGA AGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAA GAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTT ACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAAC AACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAA GATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTG CAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGG AGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAG CTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGT gggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGT CGACG

CGGCAAGAAGAAGGG TGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTG GTCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCA TCACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCAC CAGCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATC GTGTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTAT GCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAA GGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGC TGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCT CCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCG CCGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCC TACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACG CCGTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGC GGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCG GTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAA TCACCAAAATCGATAGGGAGGTT

TATCCCTACGATGTGCCCGATTACGCT GGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGA TCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGG CTACAAAGAGGCCAGCGGTTCCGGACGGGCT

ATTAACTCTA GAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGC CAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGA AGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAG CGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGC AGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCA CCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTT CCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCT CAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGT CTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACC TCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAG GGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATC TGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCT GGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATC CCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGG CCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCC TGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGAC GAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTG

SEQ ID NO: 54 is a nucleotide sequence encoding DB_(Gal4)-NS3-TA_(VPR)·DB_(Gal4) is in bold, NS4A is in italics, NS3 is in bold/underlined, NS3 Cut Site is in bold/italics, NLS is underlined, VP64 is in bold/italics/underlined, P65 in double underlined, Rta in bold, double underlined.

(SEQ ID NO: 54) ATGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGA AGAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAA GAATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTT ACGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAAC AACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAA GATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTG CAGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGG AGACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAG CTCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGT gggGCGTCTGCAggcATGGCCAGCATGACTGGTGGACAGCAAATGGGGT CGACG

CGGCAAGAAGAAGGG TGATATCGACACCTACCGATACATAGGCTCTTCCGGGACAGGCTGCGTGG TCATAGTGGGCAGGATCGTCTTGTCCGGATCCGGCACTAGT GCGCCCATC ACGGCGTACGCCCAGCAGACGAGAGGCCTCCTAGGGTGTATAATCACCA GCCTGACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATCGT GTCAACTGCTACCCAAACCTTCCTGGCAACGTGCATCAATGGGGTATGC TGGGCAGTCTACCACGGGGCCGGAACGAGGACCATCGCATCACCCAAGG GTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCTTGTGGGCTG GCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCGGCTCC TCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCC GGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTA CTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCC GTGGGCCTATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGG TGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGT GTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATC ACCAAAATCGATAGGGAGGTT

TATCCCTACGATGTGCCCGATTACGC TGGCGCGTCGTCTGCATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGG ATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAG GCTACAAAGAGGCCAGCGGTTCCGGACGGGCT

ATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAA ACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGG ATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGA AGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAG AATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCC CAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGT TCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCT GGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCA GCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTG TGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACC TACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAG TTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTG CCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCT GCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTG ATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGC CTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGG ACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTC TCAGCCTTGCTGGGCTCTGGCAGCGGCAGC

SEQ ID NO: 55 is a nucleotide sequence encoding DB_(rTetR)-NS3-TA_(VP64-p65). rTet in bold, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, NLS underlined, VP64 in bold/italics/underlined, and P65 is double underlined.

(SEQ ID NO: 55) ATGTCTAGACTGGACAAGAGCAAAGTCATAAACGGAGCTCTGGAATTAC TCAATGGTGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAA GCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGG GCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCC ACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAA CGCCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAA GTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAA ATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACT GTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAG GAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCA GGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGC CTGGAGAAACAGCTAAAGTGCGAAAGCgggGCGTCTGCAggcATGGCCA GCATGACTGGTGGACAGCAAATGGGGTCGACG

GGCAAGAAGAAGGGTGATATCGACACCTACCG ATACATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATC GTCTTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGC AGACGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGA CAAAAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAA ACCTTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACG GGGCCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGAT GTATACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGT TCCCGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGG TCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAG GGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCG GGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGG CCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGT GGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCC TCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGG AGGTT

TATCCCTACGATGTGCCCGATTACGCTGGCGCGTCGTCTGC ATGCCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGAC GCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCA GCGGTTCCGGACGGGCT

ATTAACTCTAGAAGTTCCGGATCTCCGAAAAA GAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCAC CGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCA TGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAG AAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCC CCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACG AGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGC TCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCA CCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGC CTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAA ACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTG CAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATC CTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCA GCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATG CTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGA GGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAA TGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGAT TTCTCAGCCTTGCTG

SEQ ID NO: 58 is a nucleotide sequence encoding myr-palm-NS3-Gal4_(min). Myr-palm in bold/italics/underlined, NS4A in italics, NS3 in bold/underlined, NS3 Cut Site in bold/italics, DB_(Gal4) in bold, TA_(Gal4) is double underlined.

(SEQ ID NO: 58)

CGTACGATGGCCAGCATGACTGGT GGACAGCAAATGGGGTCGACG

GGCAAGAAGAAGGGTGATATCGACACCTACCGATA CATAGGCTCTTCCGGGACAGGCTGCGTGGTCATAGTGGGCAGGATCGTC TTGTCCGGATCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGA CGAGAGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAA AAACCAAGTGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACC TTCCTGGCAACGTGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGG CCGGAACGAGGACCATCGCATCACCCAAGGGTCCTGTCATCCAGATGTA TACCAATGTGGACCAAGACCTTGTGGGCTGGCCCGCTCCTCAAGGTTCC CGCTCATTGACACCCTGTACCTGCGGCTCCTCGGACCTTTACCTGGTCA CGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGAGGTGATAGCAGGGG TAGCCTGCTTTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGG GGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCCTATTCAGGGCCG CGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATCCCTGTGGA GAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTCCTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGG TT

TATCCCTACGATGTGCCCGATTACGCTGGCGCGTCTGCATGCGGTACCA TGAAGTTGCTGAGCAGCATAGAGCAAGCATGTGATATCTGCCGGTTGAA GAAGCTGAAGTGTAGCAAGGAGAAGCCCAAGTGCGCCAAGTGTCTCAAG AATAATTGGGAGTGTAGGTATAGCCCCAAGACCAAGCGAAGCCCGCTTA CGAGAGCACACCTTACCGAGGTCGAGAGCCGCCTGGAAAGACTCGAACA ACTTTTTCTTCTGATTTTCCCCAGGGAGGACCTGGACATGATCCTGAAG ATGGACAGCCTCCAGGACATCAAAGCCCTTCTTACCGGGCTGTTCGTGC AGGACAACGTCAACAAGGATGCGGTGACCGACAGATTGGCGAGCGTGGA GACGGACATGCCCTTGACCCTCAGACAACATAGGATCAGCGCGACAAGC TCATCTGAAGAATCTAGCAATAAGGGACAGCGACAGCTGACCGTTAGTG CCAACTTTAATCAAAGTGGAAACATCGCGGACAGCTCACTCAGCTTTAC CTTCACCAATAGCAGTAACGGGCCGAACCTCATAACCACCCAGACCAAC AGCCAGGCCTTGAGCCAGCCGATCGCCTCATCTAACGTGCATGATAACT TTATGAACAACGAGATCACCGCGAGTAAGATAGACGACGGGAACAACAG CAAGCCCCTTAGCCCAGGTTGGACGGACCAGACCGCCTACAACGCTTTC GGCATTACGACCGGCATGTTCAACACCACGACCATGGACGATGTGTACA ACTACCTGTTCGATGACGAAGACACACCGCCAAACCCCAAAAAAGAA

SEQ ID NOs 62-64 are nucleotide sequences encoding sgRNAs, e.g., used herein. These sequences are further described in Zalatan et al. Cell. 2015., Nihongaki et al., Nature Chem Bio. 2015, which are incorporated herein by reference in their entireties.

sgC2¹ GCAGACGCGAGGAAGGAGGGCGC SEQ ID NO: 62 sgC3¹ GCCTCTGGGAGGTCCTGTCCGGCTC SEQ ID NO: 63 GAL4 TGGGTCTTCGGAGGACAGTACTC SEQ ID NO: 64 sgRNA1²

SEQ ID NO: 65 is a nucleotide sequence encoding NS3-Dll1-mCherry. Signal sequence in bold, Rat Dll1 ECD in italics, NS3 cut site underlined, NS4A in bold/italics, NS3 in bold/underlined, Rat Dll1 TMD/ICD in bold/italics/underlined, mCherry in double underlined.

(SEQ ID NO: 65) ATGGGCCGTCGGAGCGCTCTAGCCCTTGCCGTGGTCTCAGCCCTGCTGTGC CA GGTCTGGAGCTCTGGCGTATTTGAGCTGAAGCTGCAGGAGTTCGTCAACAAGAAGGGGCT GCTGGGGAACCGCAACTGCTGCCGCGGGGGCTCTGGCCCGCCGTGCGCCTGCAGGACC TTCTTTCGCGTATGCCTCAAGCATTACCAGGCCAGCGTGTCCCCGGAGCCACCCTGCACC TACGGCAGTGCGGTCACCGCAGTGCTGGGTGTCGACTCCTTCAGCCTGCCTGATGGCGC AGGCATCGACCCCGCCTTCAGCAACCCCATCCGATTCCCCTTCGGATTCACCTGGCCAGG TACCTTCTCTCTGATCATTGAAGCCCTCCACACAGATTCTCCTGACGACCTCGCAACAGAA AACCCAGAAAGACTCATCAGCCGCCTGACCACACAGAGGCACCTCACTGTGGGAGAAGAG TGGTCTCAGGACCTTCACAGTAGCGGCCGCACAGACCTCCGCTACTCTTACCGGTTTGTG TGTGATGAACACTACTATGGAGAAGGCTGCTCCGTGTTCTGCCGACCGCGGGATGATGCC TTTGGCCACTTCACCTGCGGGGAGAGAGGGGAGAAGATGTGCGACCCTGGCTGGAAAGG CCAGTACTGCACTGACCCCATTTGTCTGCCAGGCTGTGATGACCAACATGGATATTGTGAC AAACCGGGGGAATGCAAGTGCAGAGTTGGCTGGCAGGGCCGCTACTGCGATGAATGCAT CCGATACCCAGGCTGTCTCCATGGTACCTGCCAGCAGCCCTGGCAGTGTAACTGCCAGGA AGGCTGGGGGGGCCTCTTCTGCAACCAGGATCTGAACTACTGCACTCACCATAAGCCATG CAGGAACGGAGCCACCTGCACCAACACGGGCCAGGGGAGCTACACATGCTCTTGCCGAC CCGGGTATACAGGGGCCAACTGTGAGCTGGAGGTAGATGAGTGTGCTCCCAGCCCCTGC AGGAATGGAGGGAGCTGCACGGATCTTGAGGACAGCTACTCTTGCACCTGCCCTCCTGG CTTCTATGGCAAGGTCTGTGAGCTGAGCGCCATGACGTGTGCAGATGGTCCTTGCTTCAA TGGGGGACGATGTTCGGATAACCCCGATGGAGGCTACACCTGCCATTGCCCTGCGGGCT TCTCTGGCTTCAACTGTGAGAAGAAGATTGATCTCTGTAGCTCTTCCCCTTGTTCTAACGGT GCCAAGTGTGTGGACCTCGGCAACTCCTACCTGTGCCGATGTCAGACTGGCTTCTCCGGG AGGTACTGCGAGGACAATGTGGATGACTGTGCCTCTTCTCCCTGTGCAAACGGGGGCACC TGCCGGGACAGTGTGAACGATTTCTCCTGTACCTGCCCACCTGGCTACACAGGCAGGAAC TGCAGCGCCCCTGTCAGCAGGTGTGAGCATGCACCCTGTCATAACGGGGCCACCTGCCA CCAGAGGGGCCAACGCTACATGTGTGAGTGCGCCCAGGGCTATGGCGGCGCCAACTGCC AGTTCCTGCTCCCTGAGCCACCACCAGACCTCATAGTGGCGGCCCAGGGCGGGTCCTTC CCCTGGAGCAGGGCTGACATGGCCAGCATGACTGGTGGACAGCAAATGGGGTCGAC GGAGGACGTGGTGTGCTGCCACTCAATCTACGGCAAGAAGAAGGGTGATATCGACA CCTACCGATACATAGGCTCTTCCGGGACA

TCCGGCACTAGT GCGCCCATCACGGCGTACGCCCAGCAGACGAG AGGCCTCCTAGGGTGTATAATCACCAGCCTGACTGGCCGGGACAAAAACCAAG TGGAGGGTGAGGTCCAGATCGTGTCAACTGCTACCCAAACCTTCCTGGCAACG TGCATCAATGGGGTATGCTGGGCAGTCTACCACGGGGCCGGAACGAGGACCAT CGCATCACCCAAGGGTCCTGTCATCCAGATGTATACCAATGTGGACCAAGACCT TGTGGGCTGGCCCGCTCCTCAAGGTTCCCGCTCATTGACACCCTGTACCTGCG GCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGC CGGCGAGGTGATAGCAGGGGTAGCCTGCTTTCGCCCCGGCCCATTTCCTACTT GAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCGGGACACGCCGTGGGCC TATTCAGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAAGCGGTGGACTTTATC CCTGTGGAGAACCTAGAGACAACCATGAGATCCCCGGTGTTCACGGACAACTC CTCT CCACCAGCAGTCACCCTGACGCACCCAATCACCAAAATCGATAGGGAGGTTC TCTACCAGGAGTTCGATGAGATGGAAGAGTGCTCTCAGCACTATCCCTACGATGTGC CCGATTACGCTGGCGCGTCTGCA

GTGAGCAAGGGCGAGGAGGAT AACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTG AACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCAC CCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACA TCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACA TCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGA ACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGC GAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTA ATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGA CGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACT ACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGC GCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATC GTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCT GTACAAGTCT SEQ ID NO: 66 is a nucleotide sequence encoding hN1. (SEQ ID NO: 66) ATGCCGCCGCTCCTGGCGCCCCTGCTCTGCCTGGCGCTGCTGCCCGCGCTCGCCGCA CGAGGCCCGCGATGCTCCCAGCCCGGTGAGACCTGCCTGAATGGCGGGAAGTGTGA AGCGGCCAATGGCACGGAGGCCTGCGTCTGTGGCGGGGCCTTCGTGGGCCCGCGAT GCCAGGACCCCAACCCGTGCCTCAGCACCCCCTGCAAGAACGCCGGGACATGCCAC GTGGTGGACCGCAGAGGCGTGGCAGACTATGCCTGCAGCTGTGCCCTGGGCTTCTCT GGGCCCCTCTGCCTGACACCCCTGGACAATGCCTGCCTCACCAACCCCTGCCGCAAC GGGGGCACCTGCGACCTGCTCACGCTGACGGAGTACAAGTGCCGCTGCCCGCCCGG CTGGTCAGGGAAATCGTGCCAGCAGGCTGACCCGTGCGCCTCCAACCCCTGCGCCAA CGGTGGCCAGTGCCTGCCCTTCGAGGCCTCCTACATCTGCCACTGCCCACCCAGCTT CCATGGCCCCACCTGCCGGCAGGATGTCAACGAGTGTGGCCAGAAGCCCGGGCTTT GCCGCCACGGAGGCACCTGCCACAACGAGGTCGGCTCCTACCGCTGCGTCTGCCGCG CCACCCACACTGGCCCCAACTGCGAGCGGCCCTACGTGCCCTGCAGCCCCTCGCCCT GCCAGAACGGGGGCACCTGCCGCCCCACGGGCGACGTCACCCACGAGTGTGCCTGC CTGCCAGGCTTCACCGGCCAGAACTGTGAGGAAAATATCGACGATTGTCCAGGAAA CAACTGCAAGAACGGGGGTGCCTGTGTGGACGGCGTGAACACCTACAACTGCCGCT GCCCGCCAGAGTGGACAGGTCAGTACTGTACCGAGGATGTGGACGAGTGCCAGCTG ATGCCAAATGCCTGCCAGAACGGCGGGACCTGCCACAACACCCACGGTGGCTACAA CTGCGTGTGTGTCAACGGCTGGACTGGTGAGGACTGCAGCGAGAACATTGATGACT GTGCCAGCGCCGCCTGCTTCCACGGCGCCACCTGCCATGACCGTGTGGCCTCCTTCT ACTGCGAGTGTCCCCATGGCCGCACAGGTCTGCTGTGCCACCTCAACGACGCATGCA TCAGCAACCCCTGTAACGAGGGCTCCAACTGCGACACCAACCCTGTCAATGGCAAG GCCATCTGCACCTGCCCCTCGGGGTACACGGGCCCGGCCTGCAGCCAGGACGTGGAT GAGTGCTCGCTGGGTGCCAACCCCTGCGAGCATGCGGGCAAGTGCATCAACACGCT GGGCTCCTTCGAGTGCCAGTGTCTGCAGGGCTACACGGGCCCCCGATGCGAGATCGA CGTCAACGAGTGCGTCTCGAACCCGTGCCAGAACGACGCCACCTGCCTGGACCAGA TTGGGGAGTTCCAGTGCATCTGCATGCCCGGCTACGAGGGTGTGCACTGCGAGGTCA ACACAGACGAGTGTGCCAGCAGCCCCTGCCTGCACAATGGCCGCTGCCTGGACAAG ATCAATGAGTTCCAGTGCGAGTGCCCCACGGGCTTCACTGGGCATCTGTGCCAGTAC GATGTGGACGAGTGTGCCAGCACCCCCTGCAAGAATGGTGCCAAGTGCCTGGACGG ACCCAACACTTACACCTGTGTGTGCACGGAAGGGTACACGGGGACGCACTGCGAGG TGGACATCGATGAGTGCGACCCCGACCCCTGCCACTACGGCTCCTGCAAGGACGGC GTCGCCACCTTCACCTGCCTCTGCCGCCCAGGCTACACGGGCCACCACTGCGAGACC AACATCAACGAGTGCTCCAGCCAGCCCTGCCGCCACGGGGGCACCTGCCAGGACCG CGACAACGCCTACCTCTGCTTCTGCCTGAAGGGGACCACAGGACCCAACTGCGAGAT CAACCTGGATGACTGTGCCAGCAGCCCCTGCGACTCGGGCACCTGTCTGGACAAGAT CGATGGCTACGAGTGTGCCTGTGAGCCGGGCTACACAGGGAGCATGTGTAACATCA ACATCGATGAGTGTGCGGGCAACCCCTGCCACAACGGGGGCACCTGCGAGGACGGC ATCAATGGCTTCACCTGCCGCTGCCCCGAGGGCTACCACGACCCCACCTGCCTGTCT GAGGTCAATGAGTGCAACAGCAACCCCTGCGTCCACGGGGCCTGCCGGGACAGCCT CAACGGGTACAAGTGCGACTGTGACCCTGGGTGGAGTGGGACCAACTGTGACATCA ACAACAATGAGTGTGAATCCAACCCTTGTGTCAACGGCGGCACCTGCAAAGACATG ACCAGTGGCTACGTGTGCACCTGCCGGGAGGGCTTCAGCGGTCCCAACTGCCAGACC AACATCAACGAGTGTGCGTCCAACCCATGTCTGAACCAGGGCACGTGTATTGACGAC GTTGCCGGGTACAAGTGCAACTGCCTGCTGCCCTACACAGGTGCCACGTGTGAGGTG GTGCTGGCCCCGTGTGCCCCCAGCCCCTGCAGAAACGGCGGGGAGTGCAGGCAATC CGAGGACTATGAGAGCTTCTCCTGTGTCTGCCCCACGGGCTGGCAAGCAGGGCAGA CCTGTGAGGTCGACATCAACGAGTGCGTTCTGAGCCCGTGCCGGCACGGCGCATCCT GCCAGAACACCCACGGCGGCTACCGCTGCCACTGCCAGGCCGGCTACAGTGGGCGC AACTGCGAGACCGACATCGACGACTGCCGGCCCAACCCGTGTCACAACGGGGGCTC CTGCACAGACGGCATCAACACGGCCTTCTGCGACTGCCTGCCCGGCTTCCGGGGCAC TTTCTGTGAGGAGGACATCAACGAGTGTGCCAGTGACCCCTGCCGCAACGGGGCCA ACTGCACGGACTGCGTGGACAGCTACACGTGCACCTGCCCCGCAGGCTTCAGCGGG ATCCACTGTGAGAACAACACGCCTGACTGCACAGAGAGCTCCTGCTTCAACGGTGGC ACCTGCGTGGACGGCATCAACTCGTTCACCTGCCTGTGTCCACCCGGCTTCACGGGC AGCTACTGCCAGCACGATGTCAATGAGTGCGACTCACAGCCCTGCCTGCATGGCGGC ACCTGTCAGGACGGCTGCGGCTCCTACAGGTGCACCTGCCCCCAGGGCTACACTGGC CCCAACTGCCAGAACCTTGTGCACTGGTGTGACTCCTCGCCCTGCAAGAACGGCGGC AAATGCTGGCAGACCCACACCCAGTACCGCTGCGAGTGCCCCAGCGGCTGGACCGG CCTTTACTGCGACGTGCCCAGCGTGTCCTGTGAGGTGGCTGCGCAGCGACAAGGTGT TGACGTTGCCCGCCTGTGCCAGCATGGAGGGCTCTGTGTGGACGCGGGCAACACGC ACCACTGCCGCTGCCAGGCGGGCTACACAGGCAGCTACTGTGAGGACCTGGTGGAC GAGTGCTCACCCAGCCCCTGCCAGAACGGGGCCACCTGCACGGACTACCTGGGCGG CTACTCCTGCAAGTGCGTGGCCGGCTACCACGGGGTGAACTGCTCTGAGGAGATCGA CGAGTGCCTCTCCCACCCCTGCCAGAACGGGGGCACCTGCCTCGACCTCCCCAACAC CTACAAGTGCTCCTGCCCACGGGGCACTCAGGGTGTGCACTGTGAGATCAACGTGGA CGACTGCAATCCCCCCGTTGACCCCGTGTCCCGGAGCCCCAAGTGCTTTAACAACGG CACCTGCGTGGACCAGGTGGGCGGCTACAGCTGCACCTGCCCGCCGGGCTTCGTGGG TGAGCGCTGTGAGGGGGATGTCAACGAGTGCCTGTCCAATCCCTGCGACGCCCGTGG CACCCAGAACTGCGTGCAGCGCGTCAATGACTTCCACTGCGAGTGCCGTGCTGGTCA CACCGGGCGCCGCTGCGAGTCCGTCATCAATGGCTGCAAAGGCAAGCCCTGCAAGA ATGGGGGCACCTGCGCCGTGGCCTCCAACACCGCCCGCGGGTTCATCTGCAAGTGCC CTGCGGGCTTCGAGGGCGCCACGTGTGAGAATGACGCTCGTACCTGCGGCAGCCTGC GCTGCCTCAACGGCGGCACATGCATCTCCGGCCCGCGCAGCCCCACCTGCCTGTGCC TGGGCCCCTTCACGGGCCCCGAATGCCAGTTCCCGGCCAGCAGCCCCTGCCTGGGCG GCAACCCCTGCTACAACCAGGGGACCTGTGAGCCCACATCCGAGAGCCCCTTCTACC GTTGCCTGTGCCCCGCCAAATTCAACGGGCTCTTGTGCCACATCCTGGACTACAGCT TCGGGGGTGGGGCCGGGCGCGACATCCCCCCGCCGCTGATCGAGGAGGCGTGCGAG CTGCCCGAGTGCCAGGAGGACGCGGGCAACAAGGTCTGCAGCCTGCAGTGCAACAA CCACGCGTGCGGCTGGGACGGCGGTGACTGCTCCCTCAACTTCAATGACCCCTGGAA GAACTGCACGCAGTCTCTGCAGTGCTGGAAGTACTTCAGTGACGGCCACTGTGACAG CCAGTGCAACTCAGCCGGCTGCCTCTTCGACGGCTTTGACTGCCAGCGTGCGGAAGG CCAGTGCAACCCCCTGTACGACCAGTACTGCAAGGACCACTTCAGCGACGGGCACT GCGACCAGGGCTGCAACAGCGCGGAGTGCGAGTGGGACGGGCTGGACTGTGCGGAG CATGTACCCGAGAGGCTGGCGGCCGGCACGCTGGTGGTGGTGGTGCTGATGCCGCC GGAGCAGCTGCGCAACAGCTCCTTCCACTTCCTGCGGGAGCTCAGCCGCGTGCTGCA CACCAACGTGGTCTTCAAGCGTGACGCACACGGCCAGCAGATGATCTTCCCCTACTA CGGCCGCGAGGAGGAGCTGCGCAAGCACCCCATCAAGCGTGCCGCCGAGGGCTGGG CCGCACCTGACGCCCTGCTGGGCCAGGTGAAGGCCTCGCTGCTCCCTGGTGGCAGCG AGGGTGGGCGGCGGCGGAGGGAGCTGGACCCCATGGACGTCCGCGGCTCCATCGTC TACCTGGAGATTGACAACCGGCAGTGTGTGCAGGCCTCCTCGCAGTGCTTCCAGAGT GCCACCGACGTGGCCGCATTCCTGGGAGCGCTCGCCTCGCTGGGCAGCCTCAACATC CCCTACAAGATCGAGGCCGTGCAGAGTGAGACCGTGGAGCCGCCCCCGCCGGCGCA GCTGCACTTCATGTACGTGGCGGCGGCCGCCTTTGTGCTTCTGTTCTTCGTGGGCTGC GGGGTGCTGCTGTCCCGCAAGCGCCGGCGGCAGCATGGCCAGCTCTGGTTCCCTGAG GGCTTCAAAGTGTCTGAGGCCAGCAAGAAGAAGCGGCGGGAGCCCCTCGGCGAGGA CTCCGTGGGCCTCAAGCCCCTGAAGAACGCTTCAGACGGTGCCCTCATGGACGACAA CCAGAATGAGTGGGGGGACGAGGACCTGGAGACCAAGAAGTTCCGGTTCGAGGAGC CCGTGGTTCTGCCTGACCTGGACGACCAGACAGACCACCGGCAGTGGACTCAGCAG CACCTGGATGCCGCTGACCTGCGCATGTCTGCCATGGCCCCCACACCGCCCCAGGGT GAGGTTGACGCCGACTGCATGGACGTCAATGTCCGCGGGCCTGATGGCTTCACCCCG CTCATGATCGCCTCCTGCAGCGGGGGCGGCCTGGAGACGGGCAACAGCGAGGAAGA GGAGGACGCGCCGGCCGTCATCTCCGACTTCATCTACCAGGGCGCCAGCCTGCACAA CCAGACAGACCGCACGGGCGAGACCGCCTTGCACCTGGCCGCCCGCTACTCACGCTC TGATGCCGCCAAGCGCCTGCTGGAGGCCAGCGCAGATGCCAACATCCAGGACAACA TGGGCCGCACCCCGCTGCATGCGGCTGTGTCTGCCGACGCACAAGGTGTCTTCCAGA TCCTGATCCGGAACCGAGCCACAGACCTGGATGCCCGCATGCATGATGGCACGACG CCACTGATCCTGGCTGCCCGCCTGGCCGTGGAGGGCATGCTGGAGGACCTCATCAAC TCACACGCCGACGTCAACGCCGTAGATGACCTGGGCAAGTCCGCCCTGCACTGGGCC GCCGCCGTGAACAATGTGGATGCCGCAGTTGTGCTCCTGAAGAACGGGGCTAACAA AGATATGCAGAACAACAGGGAGGAGACACCCCTGTTTCTGGCCGCCCGGGAGGGCA GCTACGAGACCGCCAAGGTGCTGCTGGACCACTTTGCCAACCGGGACATCACGGAT CATATGGACCGCCTGCCGCGCGACATCGCACAGGAGCGCATGCATCACGACATCGT GAGGCTGCTGGACGAGTACAACCTGGTGCGCAGCCCGCAGCTGCACGGAGCCCCGC TGGGGGGCACGCCCACCCTGTCGCCCCCGCTCTGCTCGCCCAACGGCTACCTGGGCA GCCTCAAGCCCGGCGTGCAGGGCAAGAAGGTCCGCAAGCCCAGCAGCAAAGGCCTG GCCTGTGGAAGCAAGGAGGCCAAGGACCTCAAGGCACGGAGGAAGAAGTCCCAGG ACGGCAAGGGCTGCCTGCTGGACAGCTCCGGCATGCTCTCGCCCGTGGACTCCCTGG AGTCACCCCATGGCTACCTGTCAGACGTGGCCTCGCCGCCACTGCTGCCCTCCCCGT TCCAGCAGTCTCCGTCCGTGCCCCTCAACCACCTGCCTGGGATGCCCGACACCCACC TGGGCATCGGGCACCTGAACGTGGCGGCCAAGCCCGAGATGGCGGCGCTGGGTGGG GGCGGCCGGCTGGCCTTTGAGACTGGCCCACCTCGTCTCTCCCACCTGCCTGTGGCC TCTGGCACCAGCACCGTCCTGGGCTCCAGCAGCGGAGGGGCCCTGAATTTCACTGTG GGCGGGTCCACCAGTTTGAATGGTCAATGCGAGTGGCTGTCCCGGCTGCAGAGCGG CATGGTGCCGAACCAATACAACCCTCTGCGGGGGAGTGTGGCACCAGGCCCCCTGA GCACACAGGCCCCCTCCCTGCAGCATGGCATGGTAGGCCCGCTGCACAGTAGCCTTG CTGCCAGCGCCCTGTCCCAGATGATGAGCTACCAGGGCCTGCCCAGCACCCGGCTGG CCACCCAGCCTCACCTGGTGCAGACCCAGCAGGTGCAGCCACAAAACTTACAGATG CAGCAGCAGAACCTGCAGCCAGCAAACATCCAGCAGCAGCAAAGCCTGCAGCCGCC ACCACCACCACCACAGCCGCACCTTGGCGTGAGCTCAGCAGCCAGCGGCCACCTGG GCCGGAGCTTCCTGAGTGGAGAGCCGAGCCAGGCAGACGTGCAGCCACTGGGCCCC AGCAGCCTGGCGGTGCACACTATTCTGCCCCAGGAGAGCCCCGCCCTGCCCACGTCG CTGCCATCCTCGCTGGTCCCACCCGTGACCGCAGCCCAGTTCCTGACGCCCCCCTCG CAGCACAGCTACTCCTCGCCTGTGGACAACACCCCCAGCCACCAGCTACAGGTGCCT GAGCACCCCTTCCTCACCCCGTCCCCTGAGTCCCCTGACCAGTGGTCCAGCTCGTCCC CGCATTCCAACGTCTCCGACTGGTCCGAGGGCGTCTCCAGCCCTCCCACCAGCATGC AGTCCCAGATCGCCCGCATTCCGGAGGCCTTCAAGGCTAGCTAA. SEQ ID NO: 67 is a nucleotide sequence encoding myc-moxGFP-mN1TMD-GAL4-VP64. (SEQ ID NO: 67) GCCACCATGGCATTGCCCGTGACCGCCCTGCTGCTGCCACTGGCCTTGTTGCTCCACGCCGCGCG GCCAGAACAGAAGCTGATCAGCGAGGAGGATCTGACCGGTGTGAGCAAGGGCGAGGAGCTGTTCA CCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCTCCGTGCGG GGCGAGGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCAGCACCACCGGCAA GCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGAGCTTCTCCCGCT ACCCCGACCACATGAAGCGCCACGACTTCTTCAAGAGCGCCATGCCCGAAGGCTACGTCCAGGAG CGCACCATCTCCTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTTCAACTCCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGC ATCAAGGCCAACTTCAAGATCCGCCACAACGTGGAGGACGGCTCCGTGCAGCTCGCCGACCACTA CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGTCCACCC AGTCCAAGCTGTCCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTTCTGGAATTCGTGACC GCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGGGATCCCCGGTGGAGCCTCCGCTGCC CTCGCAGCTGCACCTCATGTACGTGGCAGCGGCCGCCTTCGTGCTCCTGTTCTTTGTGGGCTGTG GGGTGCTGCTGTCCCGCAAGCGCCGGCGGGGCTCGAGCATGAAGCTGCTGAGCAGCATCGAGCAG GCCTGTGACATCTGCCGGCTGAAGAAACTGAAGTGCAGCAAAGAAAAGCCCAAGTGCGCCAAGTG CCTGAAGAACAACTGGGAGTGCCGGTACAGCCCCAAGACCAAGAGAAGCCCCCTGACCAGAGCCC ACCTGACCGAGGTGGAAAGCCGGCTGGAAAGACTGGAACAGCTGTTTCTGCTGATCTTCCCACGC GAGGACCTGGACATGATCCTGAAGATGGACAGCCTGCAGGACATCAAGGCCCTGCTGACCGGCCT GTTCGTGCAGGACAACGTGAACAAGGACGCCGTGACCGACAGACTGGCCAGCGTGGAAACCGACA TGCCCCTGACCCTGCGGCAGCACAGAATCAGCGCCACCAGCAGCAGCGAGGAAAGCAGCAACAAG GGCCAGCGGCAGCTGACAGTGTCTGCTGCTGCAGGCGGAAGCGGAGGCTCTGGCGGATCTGATGC CCTGGACGACTTCGACCTGGATATGCTGGGCAGCGACGCCCTGGATGATTTTGATCTGGACATGC TGGGATCTGACGCTCTGGACGATTTCGATCTCGACATGTTGGGATCAGATGCACTGGATGACTTT GACCTGGACATGCTCGGATCATGA 

We claim:
 1. A synthetic Notch receptor protein comprising, from N-terminal to C-terminal and in covalent linkage, i. a ligand binding domain (LBD), ii. a Notch NRR (Negative Regulatory Region), iii. a transmembrane domain, and iv. an intracellular domain, wherein the Notch NRR is bound by a synthetic inhibitor protein comprising a Notch NRR-binding scFV fused to a transmembrane domain.
 2. The synthetic protein of claim 1, wherein the Notch NRR is a mutated Notch NRR.
 3. The synthetic protein of claim 2, wherein the mutated Notch NRR is mutated relative to Notch NRR1 of SEQ ID NO:
 8. 4. The synthetic protein of claim 1, wherein the transmembrane domain of the synthetic Notch receptor protein comprises the human Notch1 transmembrane domain of SEQ ID NO:
 13. 5. The synthetic protein of claim 1, wherein the scFV comprises, from N-terminal to C-terminal and in covalent linkage, a V_(H) domain, a linker domain, and a V_(L) domain.
 6. The synthetic protein of claim 1, wherein the scFV is selected from any one of SEQ ID NOs: 15-27.
 7. The synthetic protein of claim 1, further comprising a signal sequence N-terminal to the LBD.
 8. The synthetic protein of claim 1, wherein the Notch NRR is bound with high-affinity by the synthetic inhibitor protein.
 9. The synthetic protein of claim 1, wherein the transmembrane domain of the synthetic inhibitor protein comprises the human Notch1 transmembrane domain of SEQ ID NO:
 13. 10. The protein of claim 1, wherein the transmembrane domain fused to the Notch NRR-binding scFV is the transmembrane domain of (iii).
 11. The protein of claim 1, wherein the synthetic inhibitor protein and the synthetic Notch receptor protein are covalently linked.
 12. The protein of claim 10, wherein the synthetic inhibitor protein and the synthetic Notch receptor protein that are covalently linked comprise from N-terminal to C-terminal and in covalent linkage: i. the ligand binding domain (LBD), ii. the Notch NRR-binding scFV, iii. the Notch NRR (Negative Regulatory Region), iv. the transmembrane domain, and v. the intracellular domain, wherein the Notch NRR is bound by the Notch NRR-binding scFV.
 13. The synthetic protein of claim 12, wherein the Notch NRR is a mutated Notch NRR.
 14. The synthetic protein of claim 12, wherein the Notch NRR is a wild-type Notch NRR.
 15. A synthetic, drug-dependent protein comprising a ligand binding domain (LBD), an NS3 protease domain, and a transmembrane domain, and wherein the NS3 is located in between the protease domain. 